Dry blend formulation of tetrahydrobiopterin

ABSTRACT

Dry blend powder formulations comprising a pharmaceutical formulation containing tetrahydrobiopterin, and methods of making and using the same, are disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/666,697, filed Nov. 1, 2012; which claims the benefit of the priorityof U.S. Provisional Application Nos. 61/554,665, filed Nov. 2, 2011; and61/622,417, filed Apr. 10, 2012; the disclosure of each of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

This disclosure is generally directed to dry blend, powder formulationsand dosage forms of tetrahydrobiopterin. In particular, provided is adry blending process for tetrahydrobiopterin (i.e., BH4dihydrochloride), the powder of which can be stably packaged in sachets,stable capsule dosage forms containing a pullulan-free capsule shell, ordissolved into a stable solution and stored in hermetically andnon-hermetically sealed containers.

2. Background

Tetrahydrobiopterin (also referred to as BH4 or sapropterin) is abiogenic amine of the naturally occurring pterin family and is acofactor for a number of different enzymes, including phenylalaninehydroxylase (PAH), tyrosine 3-hydroxylase, tryptophan 5-hydroxylase, andnitric oxide synthase (NOS). Accordingly, BH4 is involved in thesynthesis of the amino acids phenylalanine, tyrosine and tryptophan, andthe neurotransmitters dopamine and serotonin. Moreover, BH4 is essentialfor NOS-catalyzed oxidation of L-arginine to L-citrulline and nitricoxide. Pterins are present in physiological fluids and tissues inreduced and oxidized forms, but only the 5,6,7,8-tetrahydrobiopterin isbiologically active. More specifically, it is the 6R enantiomer of BH4that is known to be the biologically active enantiomer. For a detailedreview of the synthesis of and disorders associated with BH4, see Blauet al., “Disorders of tetrahydrobiopterin and related biogenic amines”in C. R. Scriver et al., eds., The Metabolic and Molecular Bases ofInherited Disease, 8^(th) ed., pp. 1275-1776, McGraw-Hill (New York,2001).

Tetrahydrobiopterin is unstable, readily undergoes aerobic oxidation atroom temperature, and has a shelf-life of less than 8 hours at roomtemperature in aqueous solutions. Due to the instability of BH4, mosttetrahydrobiopterin products available on the market need to bespecially packaged or kept frozen. The instability of such BH4compositions is undesirable, and significant degradation resulting fromimproper storage could hinder therapy of patients. One example of astable BH4 tablet composition is disclosed in WO 2006/055511, which isincorporated herein by reference in its entirety. Such a tabletcomposition is sold under the tradename KUVAN®. There is a need foradditional formulations of BH4 suitable for pharmaceutical use.

SUMMARY

Provided herein is a stable dry blend formulation of tetrahydrobiopterin(BH4) or a BH4-related compound, stable dosages in the form of capsuledosages or dry powder sachet dosages, and therapeutic methods using suchdosage forms.

In one aspect of the disclosure, provided herein is a stable dry blendformation of BH4 or a BH4-related compound. In one embodiment, theformulation comprises a dry blend of BH4 or a BH4-related compound, aflavor enhancer, a sweetener, and one or more fillers wherein thecomponents are blended together. In certain embodiments, the blendingcomprises blending the fillers with the BH4 or BH4 compound and flavorenhancer in a blender to achieve a adequate mixture, further blending aportion of the blended mixture with acesulfame potassium or sucralose, aflavoring agent, and ascorbic acid and thereafter passing that mixturethrough a suitable sieve, and lastly blending the second mixture withthe remainder of the first mixture until the blend is homogenous.

In certain embodiments, the BH4 or a BH4-related compound is(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin dihydrochloride (i.e., “BH4dihydrochloride” or “sapropterin dihydrochloride”).

In certain embodiments for example, the fillers are isomalt, lactitol,maltitol, mannitol, sorbitol, xylitol, sucrose, fructose, orcombinations thereof.

In certain embodiments, for example, the dry blend formulation alsocontains a sweetener wherein the sweetener is acesulfame potassium,isomalt, Magna Sweet, maltitol, mannitol, sorbitol, sucralose, xylitol,alitmae, neohesperidin dihydrochalcone, trehalose, tagatose, neotame,saccharin and salts thereof, stevioside, erythritol, isomaltulose,polydextrose, luo han guo, monatin, cyclamate, osladine, sucrose,fructose, or glucose or combinations thereof.

In certain embodiments, for example, the flavor enhancer is anhydrouscitric acid, citric acid monohydrate, malic acid, tartic acid, sodiumcitrate, potassium citrate monohydrate, potassium citrate anhydrous, orsodium potassium tartate, ascorbic acid, sodium ascorbate, orcombinations thereof.

In certain embodiments, for example, the flavoring agent is a cherry,grape, orange, pink lemonade, raspberry, grape, lemon, orange,strawberry, tutti-frutti, tangerine, apple, watermelon, pineapple,banana, peach, kiwi, mango, mixed berry, raspberry lemonade, wildblackberry, blue raspberry, citrus, blueberry, lime, lemon lime,grapefruit, pomegranate, pear, or plum flavors, bubble gum, orcombinations thereof.

In certain embodiments, for example, the sieve is a 20 mesh sieve.

In another embodiment, the dry blend formulation is dissolved in anaqueous solution, flushed with an inert gas, and hermetically sealedwherein the active solution remains stable.

In another aspect of the disclosure, stable BH4 or BH4-related compoundscan optionally include one or more other therapeutic agents suitable forthe condition to be treated. In one embodiment, the other therapeuticagents are selected from folates, including but not limited to folateprecursors, folic acids, and folate derivatives, e.g., folinic acid(leucovorin); vitamins, such as vitamin C (ascorbic acid), vitamin B2(riboflavin), and vitamin B12; neurotransmitter precursors, such asL-dopa, carbidopa, and serotonin; 5-hydroxytryptophan; arginine; andcombinations thereof.

In another aspect of the disclosure, provided herein are therapeuticmethods using the stable dosage forms described herein. The stabledosage forms are useful for treating, ameliorating, or preventing anyBH4-responsive conditions or disorders, e.g., metabolic disordersinvolving amino acid metabolism. In one embodiment, the stable dosageforms are used to treat subjects exhibiting elevated phenylalaninelevels or decreased tyrosine levels, e.g., subjects suffering fromhyperphenylalanemia, mild phenylketonuria (PKU), or classic severe PKU.In another embodiment, the stable dosages are used to treat subjectssuffering from conditions or disorders that would benefit fromenhancement of nitric oxide synthase activity, including, but notlimited to, vascular diseases, ischemic or inflammatory diseases,diabetes, and insulin resistance. The total dose of BH4 or BH4-relatedcompound required can be administered in multiple doses or in a singledose. The dosage forms can be administered daily or at some otherinterval, e.g., every alternative day or weekly.

In another aspect of the disclosure, the stable dosage forms can be usedfor treating or ameliorating autism. In one embodiment, the BH4 orBH4-related compound can be used for treating or ameliorating autism inchildren. In one embodiment, the BH4 or BH4-related compound can be usedfor treating or ameliorating autism in adults. In a particularembodiment, the BH4 or BH4-related compound can be administered inconjunction with a second pharmaceutical composition to treat orameliorate the symptoms of autism. In a particular embodiment, thesecond pharmaceutical compound for the combination treatment can beselected from groups consisting of stimulants, antidepressants,antianxiety medications, non-stimulant ADHD medications, antipsychotics,mood stabilizers, or Alzheimer's medications.

In another aspect of the disclosure, the stable dosage forms can be usedalone or in conjunction with other therapies suitable for treating theparticular condition or disorder, including the underlying disease orclinical symptoms thereof. For example, for treatment of PKU or BH4deficiency, the dosage forms disclosed herein can be administered incombination with a protein-restricted diet, e.g., where the subject islimited to about 600 mg or less, or about 300 mg or less of proteindaily, and the subject optionally is given supplements of amino acids,such as tyrosine, valine, tryptophan, isoleucine, and/or leucine. Thedosage forms can also be administered in combination with folates,arginine, vitamins, or neurotransmitter precursors, or combinationsthereof. As another example, for vascular diseases, diabetes, or insulinresistance, the dosage forms described herein can be administered inconjunction with other therapeutic agent(s), such as anti-hypertensiveagents, anti-platelet agents, cholesterol-lowering agents, insulin, ororal hypoglycemic agents.

In another aspect of the disclosure, provided herein are stable capsuledosage forms, which comprise a pharmaceutical formulation containing BH4or a BH4-related compound that maintains its stability for an extendedperiod of time. In one embodiment, the formulation comprises acrystalline (e.g., powder) form of BH4 that is stable at roomtemperature for more than 8 hours, and a pharmaceutically acceptablecarrier, diluent, or excipient. In certain embodiments, the stablecapsules provided herein, in which BH4 exhibits unexpected stability,have a projected shelf-life of at least 2 years at room temperature. Inanother embodiment, the formulation comprises at least about 40% of BH4or a BH4-related compound by weight of the formulation.

In a further embodiment, the stable capsule dosage forms comprise apharmaceutical formulation containing BH4 or a BH4-related compound andone or more pharmaceutically acceptable excipients. In one embodiment,the excipients are selected from binders, fillers, diluents,disintegrants, glidants, acidic antioxidants, lubricants, andcombinations thereof. In certain embodiments, the formulation includesmannitol, crospovidone, ascorbic acid, sodium stearyl fumarate, andsilicon dioxide. In another embodiment, the formulation containsmicrocrystalline cellulose.

In another embodiment, the stable capsule dosage forms are storedwithout desiccant wherein the BH4 or BH4-related compound remains activewithin the stable capsules in the absence of desiccant.

In another aspect of the disclosure, provided herein are stable sachetdosage forms, which comprise a pharmaceutical formulation containing BH4or a BH4-related compound that maintains its stability for an extendedperiod of time. Sachet, for example, refers to a small bag or packetwherein the small bag or packet contains the BH4 or BH4-related compounddosage. Sachets are well known in the art and one of ordinary skill willunderstand the full breadth of the term.

In one embodiment, the sachet is a single chamber sachet. In anotherembodiment, the sachet is a double stick/dual chamber sachet wherein thedual chamber stick pack separates incompatible powder components (e.g.,Twin Stick Dual Chamber Stick Pack Design from Packing Technologies andInspection, LLC). In another embodiment, one chamber contains the activeBH4 or a BH4-related compound dry blend and the other contains the dryflavor blend. In another embodiment, the powders from both chambers canbe mixed and diluted with a liquid prior to oral ingestion.

In another embodiment, the BH4 or BH4-related compound dry powder blendis dissolved in an aqueous solution prior to ingestion. In a specificembodiment, the dry blend powder is clear when dissolved (i.e., thesolution is not cloudy).

In another embodiment, the dual chamber sachet is packaged in foilpouches. In another embodiment the BH4 or BH4-related compound is stablefor at least 3 months at room temperature. In another embodiment, theprojected shelf-life is at least 2 years at room temperature.

In further embodiments, the dual chamber sachets comprise apharmaceutical formulation containing BH4 or a BH4-related compound andone or more pharmaceutically acceptable excipients. In one embodiment,the excipients are selected from flavor enhancers, flavoring agents,sweeteners, fillers, diluents, glidants, anti-oxidants, and combinationsthereof. In one specific embodiment, the excipients can improvestability and manufacturability of the dry blend. In other embodiments,the fillers may be selected from the non-exclusive list consisting of,for example, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol,sucrose, and fructose. For the compositions and methods describedherein, particular features of the disclosure, such as components,ranges thereof, in compositions, conditions and steps, can be selectedfrom the various embodiments and examples described herein.

In another embodiment, such methods involve administering BH4, whetherswallowed as a solid or semisolid dosage form, or dissolved in a liquid,with food, e.g., a high-fat food or a high-fat and/or high-calorie meal.In another embodiment, BH4, whether swallowed or dissolved, isadministered at a specified time including but not limited to morning,day, night, same time of the day, with food, e.g., a high-fat food or ahigh-fat and/or high-calorie meal, one or more times a day. In anotherembodiment, BH4 is ingested once daily as a solid dosage form just aftermeals. In another embodiment the solid dosage form is a formulatedtablet or capsule. In more exemplary embodiments, BH4 is ingested withinapproximately 0 to 30 minutes, or 5 to 20 minutes, of eating a meal.

Other features and advantages of the disclosure will become apparentfrom the following detailed description. It should be understood,however, that the detailed description and the specific examples, whileindicating particular embodiments of the disclosure, are given by way ofillustration only, and various changes and modifications within thespirit and scope of the disclosure will become apparent to those skilledin the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the characteristic X-ray diffraction patternexhibited by polymorphic form B of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIGS. 2 and 3 show dissolution profiles (the rate of dissolution of BH4dihydrochloride from the solid dosage forms according to U.S.P. MethodII at 50 r.p.m. in 0.1 N hydrochloric acid at 37° C.) after the HPMC andgelatin capsules have been stored for various periods of time at 40° C.and about 75% relative humidity. Polymorph B of BH4 dihydrochloride wasused in both studies.

FIG. 4 compares the stability of BH4 capsules stored in high-densitypolyethylene (HDPE) bottles or HDPE bottles sealed in foil pouches at40° C. and 75% relative humidity to the stability of BH4 tablets storedin HDPE bottles or foil blister cards under the same conditions.Polymorph B of BH4 dihydrochloride was used in all of these studies.

FIG. 5 depicts the dissolution profile of 160 mg BH4 dihydrochloride(polymorph B) contained in HPMC capsules after the capsules have beenstored for 1 month at 25° C. and about 60% relative humidity, andalternatively at 40° C. and about 75% relative humidity.

FIG. 6 illustrates the dissolution profile of 200 mg BH4 dihydrochloride(polymorph B) contained in HPMC capsules after the capsules have beenstored for 3 months at 40° C. and about 75% relative humidity.

FIG. 7 displays the dissolution profile of 200 mg BH4 dihydrochloride(polymorph B) contained in HPMC capsules after the capsules have beenstored in HDPE bottles containing varying amounts of silica geldesiccant for 3 months at 40° C. and about 75% relative humidity.

FIG. 8 shows the dissolution profile of 250 mg BH4 dihydrochloride(polymorph B) contained in HPMC capsules after the capsules have beenstored for 1 month at 25° C. and about 60% relative humidity, andalternatively at 40° C. and about 75% relative humidity.

FIG. 9 shows the appearance of hydroxypropyl methylcellulose (“HPMC”)capsule contents after 6 months of storage at 40° C. and 5% relativehumidity (“RH”).

DETAILED DESCRIPTION

A first aspect of the disclosure is a stable, dry blend formulation ofBH4 or a BH4-related compound. In one embodiment, the stable dry blendformulation is achieved by mixing a BH4 or BH4-related compound with afiller, flavoring agent, and flavor enhancer in a blender and passedthrough a sieve. In certain embodiments, a filler, a BH4 or BH4-relatedcompound, and flavoring agent is blended together first, thereafter aportion of the mixture is further blended with acesulfame potassium orsucralose, a flavoring agent, and ascorbic acid, then passed through asuitable sieve, mixed with the remaining portion of the initial blend,and then blended until the mixture is homogenous.

Another aspect of the disclosure are stable dosage forms that maintainthe stability of a hygroscopic, moisture-sensitive active ingredientover time. In one embodiment, the active BH4 or a BH4-related compoundis in the form of an anhydrous polymorph of(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin dihydrochloride (i.e., “BH4dihydrochloride” or “sapropterin dihydrochloride”) that is stable atroom temperature to atmospheric oxygen and normal humidity, describedherein as polymorph B. Under certain conditions in which moisture ispresent (e.g., percent relative humidity above about 80% or amoisture-containing dosage form), polymorph B begins to absorb water,and thereby loses its crystalline form (i.e., becomes amorphous) andbecomes labile to oxidation. BH4 dihydrochloride is hygroscopic.Following moisture absorption or dissolution in the presence of water,BH4 dihydrochloride becomes labile to oxidation. The main oxidativedegradation product of BH4 is dihydrobiopterin (BH2), which in turn getsoxidized to biopterin.

Another aspect of the disclosure are stable dosage forms made from dryblending wherein the dry blending does not expose the active BH4 orBH4-related compound to moisture. Compared to wet blending formulationswhich can degrade the stable crystalline polymorph B formulation, dryblending can result in less degradation. Furthermore, the activepharmaceutical ingredient, i.e., the BH4 or BH4-related compound, of thedry blend powder retains its crystalline form throughout the dryblending process, in contrast to state changes resulting from a wetblending process.

Tetrahydrobiopterin dihydrochloride would be expected to exhibitdecreased stability if formulated in capsules. However, conventionalcapsule shells contain some amount of water. For example, capsule shellsmade of gelatin typically contain around 10 weight % water, and capsuleshells made of hydroxypropyl methylcellulose (HPMC) typically containabout 4 to 6 weight % water. Gelatin capsules are expected to transfermoisture from the capsule shell to the formulation loaded into thecapsule, particularly if the formulation contains a hygroscopicingredient (see, e.g., R. Chang et al., J. Pharm. Sci., 87: 556-558(1998)). The loss of water from the capsule shell can cause the gelatincapsules to become brittle and fracture easily (Chang et al., id.). Moreimportantly, US 2004/0043064 teaches that the moisture transmitted fromthe gelatin capsule shell to the formulation “result[s] in any number ofproblems, including degradation” of the active ingredient and reductionof its shelf-life. Moreover, US 2004/0043064 teaches that capsules madeof “a cellulose-based ether, such as hydroxypropyl methylcellulose, . .. still allow for the permeation of moisture,” and thus “are less thanoptimal since moisture transmission is often not sufficiently reduced.”

For hygroscopic, moisture-sensitive active ingredients such as BH4dihydrochloride, a capsule dosage form normally is not recommendedbecause the active ingredient would be expected to absorb water from thematerial (e.g., gelatin or HPMC) in the capsule shell and thereby becomeunstable (e.g., labile to oxidation in the case of BH4 dihydrochloride).Contrary to conventional wisdom, stable capsule dosage forms, in whichBH4 dihydrochloride has unexpected stability and prolonged shelf-lifedespite storage of such capsules at elevated temperature and highhumidity in the absence of a desiccant, have been developed and aredisclosed herein. It is particularly unexpected that the activepharmaceutical ingredient, i.e., BH4 or a BH4-related compound, withinthe capsule remains stable when stored in the absence of desiccant.

Another aspect of the disclosure are stable sachet dosage forms. Drypowder blends of BH4 and BH4-related compounds have unexpected physicalstabilities and blend uniformities. In another embodiment, the sachet isa single chamber sachet. In another embodiment, the sachet is a dualchamber sachet. In another embodiment, the dual chamber sachet is usedto separate incompatible components. In a specific embodiment, thesachet is used to prevent moisture from contacting the dry, stable BH4or BH4-related dry blend compound.

In certain embodiments, the BH4 or a BH4-related compound in the sachetis BH4 dihydrochloride. In another particular embodiment, the sweeteneris acesulfam potassium, isomalt, Magna Sweet, maltitol, mannitol,sorbitol, sucralose, xylitol, alitmae, neohesperidin dihydrochalcone,trehalose, tagatose, neotame, saccharin and salts thereof, stevioside,erythritol, isomaltulose, polydextrose, luo han guo, monatin, cyclamate,osladine, sucrose, fructose, or glucose or combinations thereof; theflavor enhancer is anhydrous citric acid, citric acid monohydrate, malicacid, tartic acid, sodium citrate, potassium citratedehydratemonohydrate, potassium citrate anhydrous, or sodium potassiumtartate or combinations thereof; the flavoring agent is a cherry, grape,orange, pink lemonade, raspberry, grape, lemon, orange, strawberry,tutti-frutti, tangerine, apple, watermelon, pineapple, banana, peach,kiwi, mango, mixed berry, raspberry lemonade, wild blackberry, blueraspberry, citrus, blueberry, lime, lemon lime, grapefruit, pomegranate,pear, or plum flavors or combinations thereof; and the sieve is a 20mesh sieve.

In another embodiment, one chamber of the dual chamber sachet maycontain the active BH4 or a BH4-related stable powder dry blend compoundand the second chamber may contain the flavor blend which, if packagedtogether could decrease the stability and/or appearance of the activeBH4 or BH4-related compound. Accordingly, the dry powder blend of BH4dihydrochloride in a dual chamber sachet dosage has unexpected stabilityand prolonged shelf-life have been developed and are disclosed herein.

In another embodiment, the sachets are sealed in Mylar foil pouches. Inanother specific embodiment, the sachets contain a desiccant. In certainembodiments, the desiccant is, for example, a montmorillonite clay, asilica gel, an indicating silica gel, a molecular sieve, calcium oxide,calcium sulfate, activated alumina, aerogel, benzophenone, bentoniteclay, calcium chloride, calcium hydride, cobalt(II) chloride, copper(II)sulfate, lithium chloride, lithium hydride, lithium bromide, magnesium,magnesium sulfate, magnesium perchlorate, a sodium-potassium alloy,phosphorus pentachloride, phosphorus pentoxide, potassium, potassiumcarbonate, sodium, sodium chlorate, sodium chloride, sodium hydride,sodium hydroxide, sodium sulfate, sodium-benzophenone, sucrose, orsulfuric acid or any combinations thereof. In another embodiment, theMylar foil pouches contain a desiccant.

DEFINITIONS

Generally, the nomenclature used herein and the laboratory procedures inorganic chemistry, medicinal chemistry, and pharmacology describedherein are those well known and commonly employed in the art. Unlessdefined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs.

As used in the specification and the accompanying claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as singular referents, unless the context clearly dictatesotherwise.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

As used herein, the terms “BH4”, “tetrahydrobiopterin,” “sapropterin,”and “SAP” are used interchangeably and encompass(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, and polymorphs, tautomers,pharmaceutically acceptable salts, and solvates thereof, unlessexpressly indicated otherwise. The term “BH4-related compound”encompasses analogs, derivatives, prodrugs, and precursors of BH4, aswell as pharmaceutically acceptable salts of BH4, unless expresslyindicated otherwise.

The term “subject” refers to an animal, including but not limited to, amammal, such as a primate (e.g., human or monkey), cow, sheep, goat,pig, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and“patient” are used interchangeably herein in reference, e.g., to amammalian subject, such as a human subject.

The terms “treat,” “treating,” and “treatment” encompass alleviating orabrogating a condition, disorder, or disease, or one or more of thesymptoms associated with the condition, disorder, or disease, andencompass alleviating or eradicating the cause(s) of the condition,disorder, or disease itself. In one embodiment, the terms “treat,”“treating,” and “treatment” refer to administration of a compound, apharmaceutical composition, or a pharmaceutical dosage form providedherein to a subject for purposes of alleviating, abrogating, orpreventing a condition, disorder, or disease, or symptom(s) associatedtherewith, or cause(s) thereof.

The terms “prevent,” “preventing,” and “prevention” encompass delayingand/or precluding the onset of a condition, disorder, or disease, and/orits attendant symptom(s); barring a subject from acquiring a disease;and reducing a subject's risk of acquiring a condition, disorder, ordisease.

The term “therapeutically effective amount” encompasses the amount of acompound that, when administered, is sufficient to prevent developmentof, or alleviate to some extent, one or more of the symptoms of thecondition, disorder, or disease being treated. The term “therapeuticallyeffective amount” also encompasses the amount of a compound that issufficient to elicit the biological or medical response of a cell,tissue, system, animal, or human, which is being sought by a researcher,veterinarian, medical doctor, or clinician.

In an embodiment, each component of a pharmaceutical formulation is“pharmaceutically acceptable” in the sense of being compatible with theother ingredients of the formulation, and being suitable for use incontact with cells, tissues, or organs of animals or humans withoutexcessive toxicity, irritation, allergic response, immunogenicity, orother adverse reactions, in the amount used in the dosage form accordingto the dosing schedule, and commensurate with a reasonable benefit/riskratio.

The terms “pharmaceutically acceptable carrier” and “pharmaceuticallyacceptable excipient” encompass pharmaceutically acceptable materials,compositions, and vehicles, such as liquid fillers, solid fillers,diluents, excipients, solvents, and encapsulating materials. Excipientsalso include all pharmaceutically acceptable dispersion media, coatings,isotonic agents, absorption delaying agents, antimicrobial agents,antibacterial agents, antifungal agents, adjuvants, and so on. The useof such media and agents in pharmaceutical formulations is well known inthe art. Except insofar as any conventional carrier or excipient isincompatible with the active ingredient, the present disclosureencompasses the use of conventional carriers and excipients in theformulations and dosage forms described herein. See, e.g., Remington:The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams &Wilkins (Philadelphia, Pa., 2005); Handbook of PharmaceuticalExcipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and theAmerican Pharmaceutical Association (2005); Handbook of PharmaceuticalAdditives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); andPharmaceutical Preformulation and Formulation, Gibson, Ed., CRC PressLLC (Boca Raton, Fla., 2004).

The term “dry blend powder” encompasses powders formed from granuleswithout moisture. A “dry blend powder” is a powder that is a homogenousblend suitable for automated sachet filling, stable in anon-hermetically sealed container, and able to dissolve in an aqueousliquid to produce a clear and/or transparent solution. For clarity, a“dry blend powder” is distinct from a wet blending product (i.e., wetmilling) because the active pharmaceutical ingredient (“API”) retainsits crystalline form throughout the dry blend manufacturing processwhereas wet blending results in loss of crystalline form of the API,particularly water soluble APIs such as BH4 or a BH4-related compound,during the manufacturing process.

The term “stable” encompasses a pharmaceutical compound or compositionthat retains greater than or equal to 90% of its initial potency after 3months of storage at 40° C. and 75% Relative Humidity (“RH”).

The term “dry blend flavor” encompasses a powder blend prepared bymixing dry ingredients of sapropterin dihydrochloride, sweetener,flavoring agent and flavor enhancer to form a uniform blend. The dryflavor blend quickly dissolves in water without heat and improves thepalatability of sapropterin dihydrochloride.

As used herein, the term “bioavailability” refers to the fraction of anadministered dose of a drug entering systemic circulation. If the drugwere administered intravenously, then its bioavailability theoreticallywould be 100%. However, if the drug were administered via other routes(such as orally), then its bioavailability would be less than 100% as aresult of, for example, incomplete absorption in the GI tract,degradation or metabolism prior to absorption, and/or hepatic first passeffect.

The term “high fat meal” refers generally to a meal of at least about700 kcal and at least about 45% fat (relative percentage of kcal whichare fat), or alternatively at least about 900 kcal and at least about50% fat. The term “high fat food” refers generally to a food comprisingat least 20 g of fat, or at least 25, 30, 35, 40, 45, or 50 g of fat,and/or at least about 45% or 50% fat. In another embodiment, a “high-fatmeal” contains fat as approximately 50% of total caloric content of themeal. In another embodiment, a “high-calorie meal” containsapproximately 800 to 1000 calories. In certain embodiments, a high-fatand high-calorie meal is used as a test meal for food-effectbioavailability and fed bioequivalence studies. This test meal mayderive approximately 150, 250, and 500-600 calories from protein,carbohydrate and fat, respectively. An example test meal consists of twoeggs fried in butter, two strips of bacon, four ounces of hash brownpotatoes and eight ounces of whole milk. Substitution is possible if asimilar amount of calories from protein, carbohydrate, and fat hascomparable meal volume and viscosity (Guidance for Industry, Food-EffectBioavailability and Fed Bioequivalence Studies, U.S. Department ofHealth and Human Services, Food and Drug Administration, Center for DrugEvaluation and Research (CDER), December 2002).

Synthesis of BH4 and BH4-Related Compounds

A variety of methods is known in the art for synthesis oftetrahydrobiopterins and precursors, derivatives, and analogs thereof.The following publications describe methods of making dihydrobiopterins,BH4 and derivatives thereof which can be used for the presentdisclosure: U.S. Pat. Nos. 2,601,215; 3,505,329; 4,540,783; 4,550,109;4,587,340; 4,595,752; 4,649,197; 4,665,182; 4,701,455; 4,713,454;4,937,342; 5,037,981; 5,198,547; 5,350,851; 5,401,844; and 5,698,408;Canadian Application Publication No. CA 2,420,374; European ApplicationNos. EP 079574 and EP 191335; Suntory Japanese Patent Publications Nos.JP 4-082888, JP 59-021685, and JP 9-157270; Sugimoto and Matsuura, Bull.Chem. Soc. Jp., 48(12): 3767-3768 (1975); Sugimoto and Matsuura, Bull.Chem. Soc. Jp., 52(1): 181-183 (1979); Matsuura et al., Chem. Lett. Jp.,735-738 (1984); Matsuura et al., Heterocycles, 23(12): 3115-3120 (1985);and Whiteley et al., Anal. Biochem., 137(2): 394-396 (1984), each ofwhich is incorporated herein by reference in its entirety. The followingpublications describe methods of synthesizing BH4 which can be used forthe present disclosure: WO 2005/049614; U.S. Pat. No. 4,540,783;Japanese Patent No. 59-021685; Schircks et al., Helv. Chim. Acta, 60:211 (1977); Sugimoto et al., Bull. Chem. Soc. Jp., 52(1): 181 (1979);Sugimoto et al., Bull. Chem. Soc. Jp., 48(12): 3767 (1975); Viscontiniet al., Helv. Chim. Acta, 52: 1225 (1969); and Matsuura et al., Chem.Lett., 735 (1984), each of which is incorporated herein by reference inits entirety.

Non-limiting examples of analogs of BH4 that can be used in theformulations, stable capsules, and methods described herein includepteridine, pterin, neopterin, biopterin, 7,8-dihydrobiopterin,6-methyltetrahydropterin, other 6-substituted tetrahydropterins,sepiapterin, 6,7-dimethyltetrahydropterin, 6-methyl biopterin, other6-substituted biopterins, and other analogs that are described in theart. Non-limiting examples of derivatives of BH4 that can be used in theformulations, stable capsules, and methods described herein include thederivatives described in U.S. Pat. Nos. 2,541,717; 2,603,643; 2,955,110;4,371,514; 4,758,571; 4,774,244; 5,902,810 and 6,162,806, each of whichis incorporated herein by reference in its entirety.

Any of the methods disclosed in the aforementioned publications or othersuitable methods can be used to produce BH4, or precursors, derivatives,or analogs thereof, for use in the dosage forms and therapeutic methodsdescribed herein.

Crystalline Polymorphs of (6R)-Tetrahydrobiopterin Hydrochloride Salt

BH4, and in particular the dihydrochloride salt of BH4, exhibitscrystalline polymorphism. The structure of(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin is:

The (6R) form of BH4 is the known biologically active form.

BH4 is unstable at ambient temperature and difficult to handle. Thedihydrochloride salt of BH4 is known to be more stable and easier tohandle than the free base (U.S. Application Publication No.2006/0035900, which is incorporated herein by reference in itsentirety). Results obtained during development of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride indicated that thecompound can exist in different crystalline forms, including polymorphicforms and solvates.

The crystalline polymorph of BH4 dihydrochloride that has been found tobe the most stable is referred to herein as “form B”, or “polymorph B”.Polymorph B is a slightly hygroscopic anhydrate with the highestthermodynamic stability, above about 20° C. Furthermore, form B can beeasily processed and handled due to its thermal stability and highmelting point, near 260° C. (ΔH_(f)>140 J/g). These properties renderpolymorph form B suitable for pharmaceutical formulations, which may beprepared at elevated temperatures. Polymorph B can be obtained as a finepowder with a particle size that may range from 0.2 μm to 500 μm.

Form B of BH4 dihydrochloride exhibits an X-ray powder diffractionpattern having peaks at, expressed in d-values (Å): 8.7 (vs), 6.9 (w),5.90 (vw), 5.63 (m), 5.07 (m), 4.76 (m), 4.40 (m), 4.15 (w), 4.00 (s),3.95 (m), 3.52 (m), 3.44 (w), 3.32 (m), 3.23 (s), 3.17 (w), 3.11 (vs),3.06 (w), 2.99 (w), 2.96 (w), 2.94 (m), 2.87 (w), 2.84 (s), 2.82 (m),2.69 (w), 2.59 (w), 2.44 (w). FIG. 1 is a graph of the characteristicX-ray powder diffraction pattern exhibited by form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride. As used herein, thefollowing abbreviations in brackets mean: (vs)=very strong intensity,(s)=strong intensity, (m)=medium intensity, (w)=weak intensity, and(vw)=very weak intensity.

All crystalline forms (including polymorphs, hydrates, and solvates),including form B, can be used for the preparation of polymorph B. Forexample, polymorph B can be obtained by phase equilibration ofsuspensions of amorphous or polymorphic forms of BH4, such as polymorphA, in suitable polar and non aqueous solvents. In one embodiment, thepharmaceutical preparations described herein comprise polymorph form Bof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Other forms of BH4 can be converted to form B in other ways, e.g., bydispersing the other form of BH4 in a solvent at room temperature,stirring the suspension at ambient temperature for a time sufficient toproduce polymorph form B, thereafter isolating crystalline form B, andremoving the solvent from the isolated form B. Ambient temperature, asused herein, means temperature in a range from about 0° C. to about 60°C., e.g., from about 15° C. to about 40° C. The applied temperature canbe changed during the preparation process by decreasing or increasingthe temperature stepwise or continuously. Suitable solvents for theconversion of other forms to form B include, but are not limited to,methanol, ethanol, isopropanol, other C₃- and C₄-alcohols, acetic acid,acetonitrile, tetrahydrofuran, methyl-t-butyl ether, 1,4-dioxane, ethylacetate, isopropyl acetate, other C₃-C₆-acetates, methyl ethyl ketone,and other methyl C₃-C₅ alkyl ketones. The time to complete phaseequilibration may be up to 30 hours, e.g., up to 20 hours or less than20 hours.

Polymorph B can also be obtained by crystallization from solventmixtures containing up to about 5% water, e.g., from mixtures ofethanol, acetic acid, and water. Polymorph B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolution, optionally at elevated temperatures, e.g., of a solid oflower energy form than form B or of form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a solvent mixturecomprising ethanol, acetic acid and water; addition of seeds to thesolution; cooling the obtained suspension; and isolation of the formedcrystals. Dissolution can be carried out at room temperature, up toabout 70° C., or up to about 50° C. The composition of the solventmixture may comprise a volume ratio of water:acetic acid:tetrahydrofuranof about 1:3:2 to about 1:9:4, e.g., about 1:5:4. The solution canoptionally be stirred. Cooling means cooling to a temperature in therange from about −40° C. to about 30° C., e.g., from about 10° C. toabout 30° C., or from about 0° C. to about 10° C. Suitable seeds arepolymorph B from another batch or crystals having a similar or identicalmorphology. After isolation, the crystalline form B can be washed with anon-solvent, such as acetone or tetrahydrofuran, and dried in the usualmanner.

Polymorph B can also be obtained by crystallization from aqueoussolutions through the addition of non-solvents, such as methanol,ethanol, and acetic acid. The crystallization and isolation procedurecan be advantageously carried out at room temperature without coolingthe solution. This process is therefore particularly suitable to becarried out in industrial scale.

In one embodiment, a composition comprising polymorph B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is prepared bydissolving form B, or a solid form other than form B, of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in water at ambienttemperature, adding a non-solvent in an amount sufficient to form asuspension, optionally stirring the suspension for a certain time, andthereafter isolating the formed crystals. The composition is furthermodified into a pharmaceutical composition as described below.

The concentration of (6R)-L-erythro-tetrahydrobiopterin dihydrochloridein the aqueous solution may be from about 10% to about 80% by weight,e.g., from about 20% to about 60% by weight, by reference to thesolution. Non-limiting examples of suitable non-solvents (solventsuseful in preparing suspensions of BH4) include methanol, ethanol, andacetic acid. The non-solvent may be added to the aqueous solution.Alternatively, the aqueous solution may be added to the non-solvent. Thestirring time after formation of the suspension may be up to 30 hours,e.g., up to 20 hours or less than 20 hours. Isolation by filtration anddrying is carried out in known manner as described above.

Polymorph B of BH4 dihydrochloride is a very stable crystalline formthat can be easily filtered off, dried, and ground to particle sizesdesired for pharmaceutical formulations. These properties make polymorphB particularly suitable for pharmaceutical application.

In one embodiment, a composition comprising polymorph B of BH4dihydrochloride is a very stable crystalline form that can be groundinto a powder for pharmaceutical formulations. This powder form can thenbe blended to make polymorph B particularly suitable for dry blendingwith excipients for pharmaceutical application.

Stable Capsule Dosage Forms

The capsule dosage forms described herein contain BH4 dihydrochloridethat exhibits unexpectedly stability. Commonly, unstable hygroscopicpharmaceuticals or pharmaceuticals unstable in the presence of waterabsorb water from the capsule walls and degrade in capsules. Withoutintending to be bound by any particular theory, one possible explanationfor the degradation (and consequent loss of efficacy) of suchpharmaceuticals is that hygroscopic pharmaceuticals placed in a capsulemay cause the capsule to become brittle and fracture as the waterpresent in the capsule shell is desorbed or leached from the capsuleshell into the filled pharmaceutical material. Typical hard gelatincapsule shells have around 10 wt. % moisture content, and typical HPMCcapsules have about 4 to 6 wt. % moisture content.

Capsule dosage forms, which comprise BH4 dihydrochloride and which arestable at room temperature or warmer for a long period of time withoutthe capsule becoming brittle and prone to fracturing and without the BH4dihydrochloride degrading, have been developed and are disclosed herein.In controlled studies, the BH4 dihydrochloride in the capsules providedherein unexpectedly displayed greater stability under acceleratedstability testing conditions than BH4 dihydrochloride contained inpreviously disclosed tablet dosage forms. In certain embodiments,greater than about 95%, greater than about 96%, greater than about 97%,greater than about 98%, greater than about 99%, greater than about99.1%, greater than about 99.2%, greater than about 99.3%, greater thanabout 99.4%, or greater than about 99.5% of the BH4 dihydrochlorideremains after a capsule filled with a formulation comprising BH4dihydrochloride is stored in a heat induction-sealed container at 40° C.and 75% relative humidity for six months. In further embodiments,greater than about 80%, greater than about 81%, greater than about 82%,greater than about 83%, greater than about 84%, greater than about 85%,greater than about 86%, greater than about 87%, greater than about 88%,greater than about 89%, greater than about 90%, greater than about 91%,greater than about 92%, greater than about 93%, greater than about 94%,or greater than about 95% of the BH4 dihydrochloride from such capsulesdissolve within 30 minutes according to U.S.P. Method II at 50 r.p.m. in0.1 N hydrochloric acid maintained at 37° C. Furthermore, the BH4 orBH4-related compound within the capsule remains stable when storedwithout desiccant.

In an embodiment, the shell of capsules comprising BH4 or a BH4-relatedcompound can comprise one or more natural, modified, or syntheticsaccharides or polysaccharides. In one embodiment, the capsule shell ismade of or contains one or more derivatives of cellulose in whichcellulose has been modified physically or chemically. In a specificembodiment, the cellulose derivative is hydroxypropyl methylcellulose(HPMC), also called “hypromellose”. In another embodiment, the capsuleshell is made of or contains one or more members of the carrageenanfamily of polysaccharides. In yet another embodiment, the capsule shellis made of or contains one or more starch derivatives in which starcheshave been modified physically or chemically.

In a further embodiment, the shell of capsules does not contain asaccharide or a polysaccharide. In a particular embodiment, the capsuleshell is made of or contains gelatin. In one embodiment, the gelatincapsule is hard gelatin capsule. In another embodiment, the gelatincapsule is not soft gelatin capsule. In a further embodiment, thecapsule shell is made of or contains gelatin and polyethylene glycol(PEG). In certain embodiments, the capsule shell is made of or containsgelatin and PEG 4000.

In another embodiment of non-polysaccharide capsules, the capsule shellis made of or contains one or more synthetic polymers. In oneembodiment, the synthetic polymers are selected from homopolymers andcopolymers formed from polyvinyl alcohol, acrylic acid, or methylmethacrylate, or combinations thereof.

The shell of capsules can also be made of or contain one or more naturalmaterials. In certain embodiments, the natural material is acacia.

When pullulan capsules, hard gelatin capsules, and HPMC capsulescomprising the same BH4-containing formulation were stored under thesame conditions, the pullulan capsule shells softened and collapsedwithin one month, unlike the other two kinds of capsules. Accordingly,in one embodiment, the shell of capsules comprising BH4 or a BH4-relatedcompound is essentially free of pullulan. As used herein, a“pullulan-free” capsule or a capsule “essentially free of pullulan” is acapsule having a shell that contains no pullulan or an amount ofpullulan such that the stability of the capsule shell is not adverselyaffected by the pullulan content. In a specific embodiment, the shell ofthe capsule does not contain pullulan. In other embodiments where thecapsule shell can contain pullulan, the capsule shell can contain nomore than about 90%, no more than about 85%, no more than about 75%, nomore than about 65%, no more than about 55%, no more than about 45%, nomore than about 35%, no more than about 25%, no more than about 15%, nomore than about 10%, no more than about 5%, no more than about 3%, nomore than about 2%, no more than about 1%, no more than about 0.5%, orno more than about 0.1% pullulan by weight.

In an embodiment, the stable capsule dosage forms comprise apharmaceutical formulation comprising a stable crystalline form of BH4or a BH4-related compound, and one or more pharmaceutically acceptableexcipients, diluents, or carriers. The capsule dosage forms optionallycan further comprise one or more other therapeutic agents useful for thecondition or disorder to be treated.

Because BH4 is more unstable in the presence of moisture, it isadvantageous to reduce or prevent the exposure of BH4 to moisture.Accordingly, in one embodiment, the excipient(s) in the pharmaceuticalformulation are anhydrous. In another embodiment, the excipient(s) arenot hygroscopic. In still another embodiment, the formulation containsone or more excipients that absorb and sequester moisture.

Excipients are well known for the various kinds of pharmaceuticalformulation known in the art and include, without limitation, binders(including natural and synthetic polymers), fillers, diluents,lubricants, glidants, surfactants, disintegration agents, sweeteningagents, flavoring agents, coloring agents, coating materials,preservatives, dyes, thickeners, adjuvants, antimicrobial agents,antioxidants, and carriers for the various kinds of formulation. In anembodiment, the pharmaceutical formulation in the capsule dosage formscomprises BH4 or a BH4-related compound and one or more pharmaceuticallyacceptable excipients selected from binders, fillers, diluents,disintegration agents, glidants, antioxidants (including acidicantioxidants), lubricants, surfactants, adjuvants, sweetening agents,flavoring agents, coloring agents, and combinations thereof. In oneembodiment, the formulation contains a binder, filler or diluent, and adisintegration agent. In another embodiment, the formulation furthercontains an acidic antioxidant. In yet another embodiment, theformulation further contains a lubricant.

Nonlimiting examples of binders useful in compositions described hereininclude natural and synthetic gums (e.g., acacia, gum tragacanth, guargum); starches and derivatives thereof (e.g., corn starch, potatostarch, pre-gelatinized starch); gelatin; alginic acid and alginates(e.g., sodium alginate); celluloses and derivatives thereof (e.g.,microcrystalline cellulose, methyl cellulose, ethyl cellulose,hydroxypropyl methyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose); biodegradablepolymers, such as homo- and co-polyesters of dicarboxylic acids,alkylene glycols, polyalkylene glycols and/or aliphatic hydroxylcarboxylic acids; homo- and co-polyamides of dicarboxylic acids,alkylene diamines, and/or aliphatic amino carboxylic acids;corresponding polyester-polyamide-co-polymers, polyanhydrides,polyorthoesters, polyphosphazene, and polycarbonates. The biologicallydegradable polymers can be linear, branched, or crosslinked. Specificexamples are poly-glycolic acid, poly-lactic acid, andpoly-d,l-lactide/glycolide. Other non-limiting examples of polymers arewater-soluble polymers, such as polyoxaalkylenes, polyoxaethylene,polyoxapropylene and mixed polymers thereof, poly-acrylamides andhydroxylalkylated polyacrylamides, poly-maleic acid and esters or amidesthereof, poly-acrylic acid and esters or amides thereof,poly-vinylalcohol und esters or ethers thereof, poly-vinylimidazole,poly-vinylpyrrolidone, and natural polymers, such as chitosan.

Non-limiting examples of fillers and diluents include talc, calciumcarbonate (e.g., granules or powder), calcium phosphate, celluloses andderivatives thereof (e.g., microcrystalline cellulose, powderedcellulose), dextrates, kaolin, mannitol, silicic acid, sorbitol, andstarches and derivatives thereof (e.g., starch, pre-gelatinized starch).

Disintegration agents are believed to assist in rapid disintegration ofsolid pharmaceuticals by absorbing water and expanding. Non-limitingexamples of disintegration agents include gums, agar, algins, alginicacid, clays, calcium carbonate, polacrilin potassium,polyvinylpyrrolidone (also called povidone), crospovidone (cross-linkedpovidone), celluloses and derivatives thereof (e.g., microcrystallinecellulose, croscarmellose sodium, cross-linked sodiumcarboxymethylcellulose (NaCMC, e.g., sold under the name AC-DI-SOL)),and starches and derivatives thereof (e.g., sodium starch glycolate,corn starch, potato starch, tapioca starch, pre-gelatinized starch).Pharmaceuticals formulated with crospovidone can exhibit more rapiddisintegration than pharmaceuticals formulated with povidone.

Antioxidants can be included in the inventive compositions and can helpstabilize tetrahydrobiopterin, especially after dissolution. Low pHaqueous solutions of BH4 are more stable than BH4 solutions of high pH.Exemplary acidic antioxidants include alpha-lipoic acid, ascorbic acid(including L-ascorbic acid, also called vitamin C), fatty acid esters ofascorbic acid such as ascorbyl palmitate and ascorbyl stearate, andsalts of ascorbic acid such as sodium, calcium, and potassium ascorbate.Non-acidic antioxidants can also be used in the dosage forms.Nonlimiting examples of non-acidic antioxidants include vitamin A(including beta-carotene and retinol), vitamin E (includingalpha-tocopherol), ebselen, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy(TEMPOL), and superoxide dismutase. Acidic additives, e.g., citric acidand malic acid, can be added to enhance stability of the dosage forms.

The amount of ascorbic acid in the dosage forms described herein canvary depending on the condition to be treated. In one embodiment, thepharmaceutical formulation in the capsule dosage forms comprisesascorbic acid in a weight ratio of ascorbic acid to BH4 of about 1:2,1:1.5, 1:1, 1.5:1 or 2:1. In another embodiment, the ascorbic acid toBH4 weight ratio in the formulation is no more than about 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, 1:19 or 1:20. In a particular embodiment, the ascorbic acid to BH4weight ratio is about 1:10 or less.

Nonlimiting examples of lubricants useful in compositions describedherein include natural and synthetic oils, fats, waxes, fatty acids, andsalts of fatty acids. Lubricants can improve processability and contentuniformity of the pharmaceutical. Non-limiting examples of lubricantsinclude mineral oil, light mineral oil, glycerin, sorbitol, mannitol,glycols (e.g., polyethylene glycol), hydrogenated vegetable oil (e.g.,peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, cornoil, soybean oil), talc, sodium lauryl sulfate, ethyl oleate, ethyllaureate, agar, various forms of silicon dioxide (e.g., syloid silicagel, coagulated aerosol of silica), stearyl fumaric acid and salt formsof stearyl fumarate (e.g., sodium stearyl fumarate), and stearic acidand salt forms of stearate (e.g., magnesium stearate, calcium stearate,zinc stearate).

Surfactants useful in compositions described herein can be anionic,cationic, amphoteric, or neutral. Nonlimiting examples of surfactantsuseful in compositions described herein include lecithin; phospholipids;alkyl sulfates, such as octyl sulfate, decyl sulfate, dodecyl sulfate,tetradecyl sulfate, hexadecyl sulfate, and octadecyl sulfate;1-acylaminoethane-2-sulfonic acids, such as1-octanoylaminoethane-2-sulfonic acid, 1-decanoylaminoethane-2-sulfonicacid, 1-dodecanoylaminoethane-2-sulfonic acid,1-tetradecanoylaminoethane-2-sulfonic acid,1-hexadecanoylaminoethane-2-sulfonic acid, and1-octadecanoylaminoethane-2-sulfonic acid; taurocholic acid andtaurodeoxycholic acid; bile acids and salts thereof, such as cholicacid, deoxycholic acid and sodium glycocholates; sodium caprate, sodiumlaurate, sodium oleate, sodium lauryl sulfate, sodium cetyl sulphate,sulfated castor oil, and sodium dioctylsulfosuccinate;cocamidopropylbetaine and laurylbetaine; fatty alcohols; cholesterols;glycerol mono- or -distearate, glycerol mono- or -dioleate, and glycerolmono- or -dipalmitate; and polyoxyethylene stearate.

Examples of sweetening agents include, without limitation, sucralose,sucrose, fructose, lactose, saccharin, sodium saccharide, and the like.Non-limiting examples of flavoring agents include peppermint, oil ofwintergreen, orange flavoring, cherry flavoring, and the like. Examplesof coloring agents include, but are not limited to, riboflavin,cochineal dye, carmine, blue No. 1 for food use, yellow No. 4 aluminumlake for food use, yellow No. 5 aluminum lake for food use, red No. 3aluminum lake for food use, red No. 106 for food use, iron sesquioxide,yellow iron sesquioxide, and the like.

The pharmaceutical formulation in the stable capsule dosage formsdescribed herein optionally can also comprise other excipients, such asmannitol, hydroxyl propyl cellulose, microcrystalline cellulose, andnon-reducing sugars, such as xylitol, sorbitol, trehalose, melezitose,planteose, and raffinose. Without intending to be bound by anyparticular theory, reducing sugars may react with BH4 under certainconditions. Other excipients useful in compositions described hereininclude phosphates, such as dicalcium phosphate.

The pharmaceutical formulation in the stable capsule dosage forms canoptionally include one or more other therapeutic agents suitable for thecondition to be treated. In one embodiment, the other therapeutic agentsare selected from folates, including, but not limited to, folateprecursors, folic acids and folate derivatives, e.g., folinic acid(leucovorin); vitamins, such as vitamin C (ascorbic acid), vitamin B2(riboflavin), and vitamin B12; neurotransmitter precursors, such asL-dopa, carbidopa, and serotonin; 5-hydroxytryptophan; arginine; andcombinations thereof.

Exemplary folates, including folate precursors, folic acids, and folatederivatives, are disclosed in U.S. Pat. Nos. 6,011,040 and 6,544,994(each of which is incorporated herein by reference in its entirety), andinclude folic acid (pteroylmonoglutamate), dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, one or more of the folylpolyglutamates, compounds in which thepyrazine ring of the pterin moiety of folic acid or of thefolylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, derivatives of all the preceding compounds in whichthe N-5 or N-10 position carries one carbon unit at various levels ofoxidation, pharmaceutically acceptable salts thereof, and combinationsof two or more thereof. Exemplary tetrahydrofolates include5-formyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methynyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5-formimino-(6S)-tetrahydrofolic acid, or (6S)-tetrahydrofolicacid, and pharmaceutically acceptable salts thereof. Exemplary saltsinclude sodium, potassium, calcium, and ammonium salts. Exemplaryrelative weight ratios of BH4 to folates to arginine can be in a rangefrom about 1:10:10 to about 10:1:1.

In a specific embodiment, the BH4 used in compositions described hereinis formulated as a dihydrochloride salt. Other salt forms of BH4possessing the desired physicochemical properties and biologicalactivity can also be used. For example, BH4 salts with inorganic ororganic acids are within the scope of the present disclosure.Nonlimiting examples of alternative BH4 salt forms include BH4 salts ofacetic acid, citric acid, oxalic acid, tartaric acid, fumaric acid, andmandelic acid. Carbonates or hydrogen carbonates are also possible.

Pharmaceutically acceptable salts can be formed with metals or amines,such as alkali and alkaline earth metals or organic amines.Pharmaceutically acceptable salts of compounds can also be prepared witha pharmaceutically acceptable cation. Suitable pharmaceuticallyacceptable cations are well known to those skilled in the art andinclude alkaline, alkaline earth, ammonium, and quaternary ammoniumcations. Non-limiting examples of metals used as cations are sodium,potassium, magnesium, ammonium, calcium, ferric, and the like.Non-limiting examples of suitable amines include isopropylamine,trimethylamine, histidine, N,N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, dicyclohexylamine, ethylenediamine,N-methylglucamine, and procaine.

Pharmaceutically acceptable salts include inorganic and organic acidsalts. Non-limiting examples of suitable salts include hydrochlorides,acetates, citrates, salicylates, nitrates, and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include those formed with, e.g., acetic, citric,oxalic, tartaric or mandelic acid, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid or phosphoric acid; with organic carboxylic,sulfonic, sulfo- or phosphor-acids or N-substituted sulfamic acids, forexample, acetic acid, phenylacetic acid, propionic acid, glycolic acid,succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid,fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid,gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylicacid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid,nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, ethane 1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,2-naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, or N-cyclohexylsulfamic acid(with the formation of cyclamates); with other acid organic compounds,such as ascorbic acid; or with amino acids, such as the 20 alpha aminoacids involved in the synthesis of proteins in nature, e.g., glutamicacid or aspartic acid.

In one embodiment, the pharmaceutical formulation in the stable capsuledosage forms comprises BH4 or a BH4-related compound, crospovidone, andstearyl fumaric acid or a salt form of stearyl fumarate. In anembodiment, the salt form of stearyl fumarate contains an alkali metalsalt, such as lithium, sodium, potassium, and cesium. In a specificembodiment, the salt form of stearyl fumarate is sodium stearylfumarate.

In an embodiment, the pharmaceutical formulation in the stable capsuledosage forms comprises an initial amount of BH4 or a BH4-relatedcompound (e.g., (6R)-L-erythro-BH4 dihydrochloride) in a range fromabout 30% to about 70%, crospovidone from about 2% to about 10%, andstearyl fumaric acid or a salt form of stearyl fumarate (e.g., sodiumstearyl fumarate) from about 0.5% to about 5%, by weight of theformulation. In narrower embodiments, the formulation comprises: (1) BH4or a BH4-related compound from about 35% to about 65%, or from about 30%to about 60%, or from about 35% to about 60%, or from about 35% to about55%, or from about 40% to about 60%, or from about 45% to about 55%; (2)crospovidone from about 2% to about 8%, or from about 3% to about 7%, orfrom about 3% to about 6%, or from about 4% to about 5%; and (3) stearylfumaric acid or a salt form of stearyl fumarate from about 1% to about4%, or from about 1% to about 3%, by weight of the formulation.

In another embodiment, the pharmaceutical formulation in the stablecapsule dosage forms further comprises an initial amount of ascorbicacid in a range from about 0% to about 50%, silicon dioxide (e.g.,colloidal silicon dioxide) from about 0% to about 5%, and mannitol fromabout 0% to about 50% by weight of the formulation. In narrowerembodiments, the formulation further comprises: (1) ascorbic acid fromabout 0.5% to about 40%, or from about 1% to about 30%, or from about 1%to about 20%, or from about 1% to about 10%, or from about 0.5% to about5%; (2) silicon dioxide from about 0.2% to about 4%, or from about 0.2%to about 3%, or from about 0.2% to about 2%; and (3) mannitol from about5% to about 50%, or from about 20% to about 50%, or from about 30% toabout 50%, or from about 25% to about 45%, or from about 10% to about40%, or from about 20% to about 40%, or from about 15% to about 35%, byweight of the formulation.

In yet another embodiment, the pharmaceutical formulation in the stablecapsule dosage forms additionally comprises an initial amount of5-hydroxytryptophan (5-HTP) in a range from about 0% to about 50% byweight of the formulation. In narrower embodiments, the formulationadditionally comprises 5-HTP from about 5% to about 45%, or from about10% to about 40%, or from about 20% to about 40%, or from about 15% toabout 35%, or from about 20% to about 30%, by weight of the formulation.

In certain embodiments, the stable capsule dosage forms described hereincontain an initial amount of BH4 or a BH4-related compound (e.g.,(6R)-L-erythro-BH4 dihydrochloride) in a range from about 100 mg toabout 500 mg per capsule, or from about 300 mg to about 500 mg percapsule, or from about 200 mg to about 400 mg per capsule, or from about100 mg to about 300 mg per capsule. In specific embodiments, the stablecapsule dosage forms contain an initial amount of BH4 or a BH4-relatedcompound of about 100 mg per capsule, or about 150 mg per capsule, orabout 160 mg per capsule, or about 200 mg per capsule, or about 250 mgper capsule, or about 300 mg per capsule, or about 350 mg per capsule,or about 400 mg per capsule, or about 450 mg per capsule, or about 500mg per capsule. It will be apparent to one skilled in the art that thedesired dosage for the treatment, amelioration or prevention of aBH4-responsive disorder can affect the amount of BH4 or a BH4-relatedcompound in the capsule dosage forms.

In one embodiment, provided herein are capsule dosage forms that containrelatively large amounts of BH4 (e.g., (6R)-L-erythro-BH4dihydrochloride). A non-limiting example of such a dosage form includes400 mg of BH4, optionally with a compaction agent, e.g.,microcrystalline cellulose. Potential advantages of relatively largeamounts of BH4 available in a dosage form include ease of providing thetherapeutic, ease of oral administration, and patient compliance.

In an embodiment, the stable capsule dosage forms described herein areuseful for treating BH4 deficiency or a condition or disorder associatedwith BH4 deficiency (e.g., hyperphenylalaninemia due to BH4 deficiency).In one embodiment, the stable capsule dosage forms useful for treatingBH4 deficiency or a condition associated therewith comprise initialamounts of (1) BH4 or a BH4-related compound (e.g., (6R)-L-erythro-BH4dihydrochloride) from about 30% to about 60%, or from about 40% to about60%; (2) mannitol from about 20% to about 50%, or from about 30% toabout 50%; (3) crospovidone from about 2% to about 8%, or from about 3%to about 6%; (4) stearyl fumaric acid or a salt form of stearyl fumarate(e.g., sodium stearyl fumarate) from about 1% to about 4%, or from about1% to about 3%; (5) ascorbic acid from about 1% to about 20%, or fromabout 1% to about 10%; and (6) silicon dioxide (e.g., colloidal silicondioxide) from about 0.2% to about 4%, or from about 0.2% to about 2%, byweight of the formulation. In a related embodiment, the stable capsulesuseful for treating BH4 deficiency or a condition associated therewithcomprise an initial amount of BH4 or a BH4-related compound (e.g.,(6R)-L-erythro-BH4 dihydrochloride) in a range from about 100 mg toabout 500 mg per capsule, including but not limited to about 100 mg,about 150 mg, about 160 mg, about 200 mg, about 250 mg, about 300 mg,about 350 mg, about 400 mg, about 450 mg, or about 500 mg per capsule.In a particular embodiment, the shell of the stable capsules useful fortreating BH4 deficiency or a condition associated therewith is made ofor comprises hydroxypropyl methylcellulose (HPMC). In anotherembodiment, the shell of such stable capsules is made of or comprisesgelatin.

In one particular embodiment, the stable capsule dosage form is usefulfor reducing blood phenylalanine (Phe) levels in patients withhyperphenylalaninemia (HPA) due to tetrahydrobiopterin (BH4) responsivePhenylketonuria (PKU). In another particular embodiment, the stablecapsule dosage is used in conjunction with a Phe-restricted diet toreduce blood phenylalanine (Phe) levels in patients withhyperphenylalaninemia (HPA) due to tetrahydrobiopterin (BH4) responsivePhenylketonuria (PKU).

In a particular embodiment of a stable capsule dosage form useful fortreating BH4 deficiency or a condition associated with BH4 deficiency(e.g., hyperphenylalaninemia due to BH4 deficiency), the pharmaceuticalformulation of the dosage form comprises the following ingredients, interms of weight % of the formulation and mg per capsule: (1) 50.0%(6R)-L-erythro-BH4 dihydrochloride (160.0 mg); (2) 38.5% mannitol (123.2mg); (3) 5.0% ascorbic acid (16.0 mg); (4) 4.0% crospovidone (12.8 mg);(5) 1.75% sodium stearyl fumarate (5.6 mg); and (6) 0.75% colloidalsilicon dioxide (2.4 mg). In one embodiment, the shell of the capsule ismade of or comprises hydroxypropyl methylcellulose. In anotherembodiment, the capsule shell is made of or comprises gelatin. In aspecific embodiment, the capsule is size 0 capsule (0.30 inch by 0.85inch).

In another particular embodiment of a stable capsule dosage form usefulfor treating BH4 deficiency or a condition associated with BH4deficiency (e.g., hyperphenylalanemia due to BH4 deficiency), thepharmaceutical formulation of the dosage form comprises the followingingredients, in terms of weight % of the formulation and mg per capsule:(1) 50.0% (6R)-L-erythro-BH4 dihydrochloride (200.0 mg); (2) 41.0%mannitol (164.0 mg); (3) 2.5% ascorbic acid (10.0 mg); (4) 4.0%crospovidone (16.0 mg); (5) 1.75% sodium stearyl fumarate (7.0 mg); and(6) 0.75% colloidal silicon dioxide (3.0 mg). In one embodiment, theshell of the capsule is made of or comprises hydroxypropylmethylcellulose. In another embodiment, the capsule shell is made of orcomprises gelatin. In an embodiment, the capsule is size 0 (0.30 inch by0.85 inch) or size 00 (0.33 inch by 0.92 inch) capsule.

In a further embodiment, the stable capsule dosage forms describedherein are useful for treating sickle cell disease (SCD), peripheralarterial disease (PAD), chronic kidney disease (CKD), or hypertension.In one embodiment, the stable capsule dosage forms useful for treatingSCD, PAD, CKD, or hypertension comprise initial amounts of (1) BH4 or aBH4-related compound (e.g., (6R)-L-erythro-BH4 dihydrochloride) fromabout 30% to about 60%, or from about 40% to about 60%, or from about40% to about 50%; (2) ascorbic acid from about 30% to about 60%, or fromabout 40% to about 60%, or from about 40% to about 50%; (3) crospovidonefrom about 2% to about 8%, or from about 3% to about 6%; (4) stearylfumaric acid or a salt form of stearyl fumarate (e.g., sodium stearylfumarate) from about 1% to about 4%, or from about 1% to about 3%; (5)silicon dioxide (e.g., colloidal silicon dioxide) from about 0.2% toabout 4%, or from about 0.2% to about 2%; and (6)5-methyltetrahydrofolate (5-MTHF) or a salt form thereof from about 0%to about 2%, or from about 0.01% to about 1%, or from about 0.01% toabout 0.5%, by weight of the formulation. The 5-MTHF can be apharmaceutically acceptable salt of 5-MTHF, e.g., the calcium salt of5-MTHF. In a related embodiment, the stable capsules useful for treatingSCD, PAD, CKD, or hypertension comprise initial amounts of BH4 or aBH4-related compound (e.g., (6R)-L-erythro-BH4 dihydrochloride) andascorbic acid in a weight ratio of about 10:1, about 9:1, about 8:1,about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2.5:1,about 2:1, about 1.5:1, about 1:1, about 1:1.5, or about 1:2. In anotherrelated embodiment, the stable capsules useful for treating SCD, PAD,CKD, or hypertension comprise an initial amount of BH4 or a BH4-relatedcompound (e.g., (6R)-L-erythro-BH4 dihydrochloride) in a range fromabout 100 mg to about 500 mg per capsule, including but not limited toabout 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg,about 350 mg, about 400 mg, about 450 mg, or about 500 mg per capsule.In a particular embodiment, the shell of the stable capsules useful fortreating SCD, PAD, CKD, or hypertension is made of or compriseshydroxypropyl methylcellulose (HPMC). In another embodiment, the shellof such stable capsules is made of or comprises gelatin.

In a particular embodiment of a stable capsule dosage form useful fortreating SCD, PAD, CKD, or hypertension, the pharmaceutical formulationof the dosage form comprises the following ingredients, in terms ofweight % of the formulation and mg per capsule: (1) 46.75%(6R)-L-erythro-BH4 dihydrochloride (250.0 mg); (2) 46.75% ascorbic acid(250.0 mg); (3) 3.98% or 3.96% crospovidone (21.3 mg or 21.2 mg); (4)1.75% sodium stearyl fumarate (9.4 mg); (5) 0.75% colloidal silicondioxide (4.0 mg); and (6) 0.02% or 0.04% 5-methyltetrahydrofolate,calcium salt (0.1 mg or 0.2 mg). In an embodiment, the shell of thecapsule is made of or comprises hydroxypropyl methylcellulose. Inanother embodiment, the capsule shell is made of or comprises gelatin.In yet another embodiment, the capsule is size 00 capsule (0.33 inch by0.92 inch).

In another particular embodiment, the stable capsule dosage form can beused for treating and ameliorating autism. BH4 has been shown to beeffective in treating autism in humans (see, e.g., Fernell et al., Dev.Med. & Child Neurology, 39: 313-318 (1997); Frye et al.Neurotherapeutics, 7: 241-249; Danfors et al., J. ClinicalPsychopharmacology, 25(5): 485-489). Fernell shows that children treatedwith BH4 for 4, 8, and 12 weeks had significant increases in their totalscores on the Parental Satisfaction Survey (“PSS”). Fernell, Table II,page 316. Frye reviews research using BH4 to treat autism and concludesthat BH4 represents a novel therapy for autism spectrum disorder.Danfors shows that children treated with BH4 had significant improvementof their social interaction scores after 6 months of treatment and founda high positive correlation between response of the social interactionscore and IQ.

In one embodiment, the BH4 or BH4-related compound can be used fortreating or ameliorating autism in children. In one embodiment, the BH4or BH4-related compound can be used for treating or ameliorating autismin adults. In a particular embodiment, the BH4 or BH4-related compoundcan be administered in conjunction with a second pharmaceuticalcomposition to treat or ameliorate the symptoms of autism. In aparticular embodiment, the second pharmaceutical compound for thecombination treatment can be selected from groups consisting ofstimulants, antidepressants, antianxiety medications, non-stimulant ADHDmedications, antipsychotics, mood stabilizers, or Alzheimer'smedications.

In a particular embodiment, the BH4 or BH4-related compound useful fortreating and ameliorating autism is BH4 dihydrochloride. In a particularembodiment, BH4 dihydrochloride can be administered to childrensuffering from autism. In particular embodiment, the child is about 0-18years old, 1-18 years old, about 2-18 years old, about 3-18 years old,about 3-17 years old, about 3-16 years old, about 3-15 years old, about3-14 years old, about 3-13 years old, about 3-12 years old, about 3-11years old, about 3-10 years old, about 3-9 years old, about 3-8 yearsold, about 3-7 years old, or about 3-6 years old. In a particularembodiment, the is administered to a subject during childhood andadulthood. In a particular embodiment, the formulation can beadministered at a dosage of about 5-50 mg/kg/day. In a particularembodiment, the formulation can be administered at a dosage of about 5mg/kg/day, or about 7.5 mg/kg/day, or about 10 mg/kg/day, or about 10.5mg/kg/day, or about 11 mg/kg/day, or about 11.5 mg/kg/day, or about 12mg/kg/day, or about 12.5 mg/kg/day, or about 13 mg/kg/day, or about 13.5mg/kg/day, or about 14 mg/kg/day, or about 14.5 mg/kg/day, or about 15mg/kg/day, or about 15.5 mg/kg/day, or about 16 mg/kg/day, or about 16.5mg/kg/day, or about 17 mg/kg/day, or about 17.5 mg/kg/day, or about 18mg/kg/day, or about 18.5 mg/kg/day, or about 19 mg/kg/day, or about 19.5mg/kg/day, or about 20 mg/kg/day. In another embodiment, thepharmaceutical formulation comprising BH4 or a BH4-related compound canbe used to treat or ameliorate autism at a dosage of 25 mg/kg/day ormore.

In a particular embodiment, the stimulants administered in conjunctionwith the BH4 or BH4-related compound to treat and ameliorate autism mayinclude but are not limited to amphetamines, methylphenidate, pemoline,dextroamphetamines, dexmethylphenidate, methylphenidate, andcombinations thereof. The non-stimulant administered in conjunction withthe BH4 or BH4-related compound to treat and ameliorate autism mayinclude but is not limited to atomoxetine. The antidepressants andanti-anxiety medications administered in conjunction with the BH4 orBH4-related compound to treat and ameliorate autism may include, but arenot limited to clomipramin, buspirone, buspirone, fluvoxamine,paroxetine, fluoxetine, nefazodone, doxepin, imipramine, bupropion,sertraline, and combinations thereof. The mood stabilizing medicationsadministered in conjunction with the BH4 or BH4-related compound totreat and ameliorate autism may include but are not limited to lithiumcitrate, valproic acid, lithium carbonate, carbamazepine, andcombinations thereof. The Alzheimer's medication administered inconjunction with the BH4 or BH4-related compound to treat and ameliorateautism may include but is not limited to memantine HCl.

Capsule dosage forms can be manufactured by admixing the pharmaceuticalcomponents, optionally finely dividing them, and filling capsules. Anadditional method for manufacturing capsule dosage forms can includecompounding tetrahydrobiopterin with an excipient, e.g., apharmaceutically acceptable carrier. Further, a capsule can then befilled with the compounded material and, optionally, the capsule can bestored in a sealed container, e.g., a heat induction-sealed polyethylenecontainer. The compounded pharmaceutical components or powder blends canbe uniformly filled into capsules using an automated capsule-fillingequipment or can be manually filled into capsules, as is known in theart. It is understood that the “initial amount” of a pharmaceuticalcomponent (e.g., BH4, a BH4-related compound, 5-hydroxytryptophan, or anexcipient) in a capsule is that amount of the component which is filledinto the capsule in the process for producing the capsule dosage form.

In an embodiment, the stable capsule dosage forms, including thepharmaceutical formulations contained therein, are made by a processthat does not include adding liquid water. The dry capsule manufacturingprocess comprises a dry process of mixing and blending an initial amountof the active ingredient, BH4 or a BH4-related compound (e.g.,(6R)-L-erythro-BH4 dihydrochloride), with an initial amount of anexcipient that optionally has been screened with an appropriate meshscreen, optionally mixing and blending the resulting ingredients with aninitial amount of a second excipient that optionally has been screened,optionally doing the same with additional excipients, and filling theresulting dry blend of ingredients into capsules.

In another embodiment, the methods involve the step of informing thepatient that absorption of tetrahydrobiopterin is increased when it isingested with food compared to when ingested without food. In someembodiments, the patient is informed that ingestion shortly following ameal, for example, a high-fat, high-calorie meal, results in an increasein any one, two, three or all of the following parameters: mean plasmaconcentration, Cmax, AUC, AUC(0-t) and/or AUC(inf). In otherembodiments, the patient is informed that administration of BH4 with ahigh-fat meal increases Cmax and AUC compared to administration of BH4without food (in a fasting condition). In some embodiments, the relativeincrease can be at least 20% or 30% or more.

In another embodiment, such methods involve administering BH4, whetherswallowed as a solid or semisolid dosage form, or dissolved in a liquid,with food, e.g., a high-fat food or a high-fat and/or high-calorie meal.In another embodiment, BH4, whether swallowed or dissolved, isadministered at a specified time including but not limited to morning,day, night, same time of the day, with food, e.g., a high-fat food or ahigh-fat and/or high-calorie meal, one or more times a day. In anotherembodiment, BH4 is ingested once daily as a solid dosage form just aftermeals. In another embodiment the solid dosage form is a formulatedtablet or capsule. In other embodiments, BH4 is ingested withinapproximately 0 to 30 minutes, or 5 to 20 minutes, of eating a meal.

The BH4 and the food may be ingested at approximately the same time, orthe BH4 may be ingested before or after the food. The period of timebetween consuming the food and taking BH4, either swallowed ordissolved, may be at least 5 minutes. For example, BH4 may beadministered 60 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes,10 minutes, or 5 minutes before or after a meal.

In another embodiment, to maximize oral bioavailability of BH4 at eachadministration, BH4 should be taken with food, e.g., a high fat food ora high fat and/or high calorie meal. Alternatively, to maximizeconsistency of oral bioavailability between administrations, BH4 shouldbe taken on an empty stomach (e.g., 1 hour before or 2 hours after ameal).

The container or packaging containing BH4 capsule dosage forms isselected, inter alia, to minimize or prevent moisture penetration intoand contamination of the formulation in the dosage form and therebyenhance the stability of the formulation. In an embodiment, BH4 capsuledosage forms are disposed in a sealed container or packaging. Examplesof sealed containers include polyethylene bottles, e.g., high densitypolyethylene bottles closed with a heat-induction seal. Additionalnon-limiting examples of sealed containers or packaging include glassbottles, tubes, hermetically sealed foil packaging, thermally sealedpolyethylene bags, laminated foil pouches, and blister packs. In oneembodiment, a desiccant is disposed in the container or packagingcontaining the BH4 capsule dosage forms, or is incorporated in theclosure, stopper, or cap of the container or packaging. In anotherembodiment, no desiccant is disposed in the container or packagingcontaining the BH4 capsule dosage forms, or is incorporated in theclosure, stopper, or cap of the container or packaging.

In another embodiment, at least about 90% of the initial amount of theBH4 or BH4-related compound remains, after the stable capsule dosageform is stored at about 40° C. and about 75% RH for a period of aboutthree months. In another embodiment, at least about 91%, or at leastabout 92%, or at least about 93%, or at least about 94%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99% of the initial amount of BH4 orBH4-related compound remains after the stable capsule dosage form isstored at about 40° C. and about 75% RH for a period of about threemonths.

In another embodiment, at least about 85% of the initial amount of theBH4 or BH4-related compound dissolves within about 15 minutes, after thestable capsule dosage form is stored at about 40° C. and about 75% RHfor a period of about three months. In another embodiment, at leastabout 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99% of the initial amount of the BH4 orBH4-related compound dissolves within about 15 minutes, after the stablecapsule dosage form is stored at about 40° C. and about 75% RH for aperiod of about three months.

In another embodiment, at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% of the initial amount of the BH4or BH4-related compound remains and at least about 85%, or at leastabout 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99% of the initial amount of the BH4 orBH4-related compound dissolves within about 15 minutes, after the stablecapsule dosage form is stored at about 40° C. and about 75% RH for aperiod of about three months.

Stable Sachet Dosage Forms

The sachet dosage forms described herein contain BH4 dihydrochloridethat exhibits unexpected physical stability. Commonly, granular forms ofBH4 dihydrochloride are made by a wet granulation process (Sugita etal., Patent Publication No. US2008/0207624 A1). The wet granulationprocess consists of mixing the active ingredients with the excipients,adding a liquid binder to the powder blend and mixing thoroughly, andthen screening the damp mass through a mesh to form granules. Wetgranulation is often preferable because a uniform and better-flowingmixture can be easily achieved.

In contrast to wet blending, the dry blend process disclosed herein doesnot require the addition of a liquid binder. Instead, the differentcomponents of the mixture are added to a blender generating a powderthrough the blending process. Thereafter the mixture can be screenedthrough a sieve and blended again to achieve homogeneity. However, notall mixtures are compatible with the dry blending process because inorder for the dry blend to form a homogonous mixture the components mustexhibit compatible abilities to flow (i.e., flowability).

Flowability refers to how a given material will flow in a given piece ofequipment. It is dependent on the multidimensional characteristics ofthe material (e.g., powder) that are not inherent. It results from acombination of the powder's physical properties (i.e., density, cohesivestrength, and wall friction) and the equipment used for handling,storing, or processing the powder. For example, powders with identicalphysical properties may flow differently through a hopper. Furthermore,flowability is a factor influenced by blending in which the quality ofthe resulting blend depends on the type of blender used and on thecomplex flow behavior of the powder during the blending cycle. This isbecause blending is accomplished by shear, convection, and diffusion andeach factor is dependent on the physical properties of the material.Accordingly, the flow properties of a blend of materials cannot bedetermined based on the flow properties of the individual components ofthe blend (e.g., the flowability of small particle mannitol is differentfrom large particle mannitol and the flow of a BH4dihydrochloride-mannitol blend is different and not predictable based onthe flow of the mannitol or BH4 alone).

Furthermore, the amount of compound used for dry blending also affectsthe flowability of the compound and mixtures thereof. Accordingly, inaddition to selecting compounds that have compatible flowcharacteristics when combined in a mixture, it is also necessary todetermine the correct concentration of BH4 dihydrochloride to include inthe dry blend powder to achieve acceptable flowability and maintain ahomogenous mixture. Thus, in order to use a dry blending process for BH4dihydrochloride, the final mixture must remain homogenous and that canonly be achieved by (1) determining the proper components of themixture, (2) the order of addition of components of mixture, and (3) bydetermining how the components of the mixture behave in conjunction withone another. Accordingly, the wet granulation process is commonly usedbecause flow properties of dry compounds are unpredictable and the wetgranulation process results in the agglomeration of dry powders intolarger and denser granules which flow better and granules remainhomogenous because of the presence of a binder or binders to preventsegregation of the granules back into its constituent excipients andactive drug substance.

However, despite the commonality of wet granulation methods, it is notideal for all blends. Specifically, BH4 dihydrochloride is moisturesensitive and will rapidly degrade in the presence of water.Furthermore, BH4 dihydrochloride is pH sensitive and commonly degradesin the presence of reducing sugars, flavoring agents, flavor enhancers,and aqueous solutions and thus wet blending of BH4 dihydrochloride withexcipients can lead to degradation of the active ingredient.Additionally, BH4 dihydrochloride which is highly soluble in water (>100mg/mL) is solubilized during the wet granulation process leading to theloss of the stable crystalline polymorph B. BH4 dihydrochlorideprecipitates as a less stable amorphous form following wet granulation.Accordingly, the dry blend method of producing a stable BH4dihydrochloride powder sachet dosage is ideal.

In one embodiment, the stable sachet dosage forms comprise apharmaceutical formulation comprising a stable crystalline (i.e.,powder) form of BH4 or a BH4-related compound, and one or morepharmaceutically acceptable excipients. The sachet dosage formsoptionally can further comprise one or more other therapeutic agentsuseful for the condition or disorder to be treated. The sachet dosageforms can further be in the form of a single chamber sachet or a dualchamber sachet. The sachet dosage forms can further contain a stable dryblend BH4 dihydrochloride powder or a stable BH4 dihydrochloridesolution that has been flushed with an inert gas and hermeticallysealed.

Because BH4 is more unstable in the presence of moisture due tooxidation, it is advantageous to reduce or prevent the exposure of BH4to moisture when the excipient and BH4 dihydrochloride are combined inthe single chamber sachet. Accordingly, in one embodiment, theexcipient(s) in the pharmaceutical formulation are anhydrous. In anotherembodiment, the excipient(s) are not hygroscopic. In another embodiment,the formulation contains one or more excipients that absorb andsequester moisture. In still another embodiment, the BH4 dihydrochloridecompound is separated from the excipient(s) that are incompatible withBH4 using a dual chamber sachet.

Excipients are well known for the various kinds of pharmaceuticalformulation known in the art and include, without limitation, binders(including natural and synthetic polymers), fillers, diluents, glidants,surfactants, sweetening agents, flavoring agents, coloring agents,coating materials, preservatives, dyes, thickeners, adjuvants,antimicrobial agents, antioxidants, and carriers for the various kindsof formulation. In an embodiment, the pharmaceutical formulation in thesachet dosage forms comprises BH4 or a BH4-related compound and one ormore pharmaceutically acceptable excipients selected from sweeteningagents, flavoring agents, flavor enhancers, and combinations thereof.

Non-limiting examples of fillers and diluents include maltose, mannitol,xylitol, sorbitol, isomalt, trehalose, and starches and derivativesthereof (e.g., starch, pre-gelatinized starch).

Antioxidants can be included in the inventive compositions and can helpstabilize tetrahydrobiopterin, especially after dissolution. Low pHaqueous solutions of BH4 are more stable than BH4 solutions of high pH.Exemplary acidic antioxidants include alpha-lipoic acid, ascorbic acid(including L-ascorbic acid, also called vitamin C), fatty acid esters ofascorbic acid such as ascorbyl palmitate and ascorbyl stearate, andsalts of ascorbic acid such as sodium, calcium, and potassium ascorbate.Non-acidic antioxidants can also be used in the dosage forms.Nonlimiting examples of non-acidic antioxidants include vitamin A(including beta-carotene and retinol), vitamin E (includingalpha-tocopherol), ebselen, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy(TEMPOL), and superoxide dismutase. Acidic additives, e.g., citric acidand malic acid, can be added to enhance stability of the dosage forms.

The amount of ascorbic acid in the dosage forms described herein canvary depending on the condition to be treated. In one embodiment, thepharmaceutical formulation in the sachet dosage forms comprises ascorbicacid in a weight ratio of ascorbic acid to BH4 of about 1:2, 1:1.5, 1:1,1.5:1 or 2:1. In another embodiment, the ascorbic acid to BH4 weightratio in the formulation is no more than about 1:2, 1:3, 1:4, 1:5, 1:6,1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19 or 1:20. In a particular embodiment, the ascorbic acid to BH4weight ratio is about 1:10 or less.

Nonlimiting examples of lubricants useful in compositions describedherein include natural and synthetic oils, fats, waxes, fatty acids, andsalts of fatty acids. Lubricants can improve processability and contentuniformity of the pharmaceutical. Non-limiting examples of lubricantsinclude mineral oil, light mineral oil, glycerin, sorbitol, mannitol,glycols (e.g., polyethylene glycol), hydrogenated vegetable oil (e.g.,peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, cornoil, soybean oil), talc, sodium lauryl sulfate, ethyl oleate, ethyllaureate, agar, various forms of silicon dioxide (e.g., syloid silicagel, coagulated aerosol of silica), stearyl fumaric acid and salt formsof stearyl fumarate (e.g., sodium stearyl fumarate), and stearic acidand salt forms of stearate (e.g., magnesium stearate, calcium stearate,zinc stearate).

Surfactants useful in compositions described herein can be anionic,cationic, amphoteric, or neutral. Nonlimiting examples of surfactantsuseful in compositions described herein include lecithin; phospholipids;alkyl sulfates, such as octyl sulfate, decyl sulfate, dodecyl sulfate,tetradecyl sulfate, hexadecyl sulfate, and octadecyl sulfate;1-acylaminoethane-2-sulfonic acids, such as1-octanoylaminoethane-2-sulfonic acid, 1-decanoylaminoethane-2-sulfonicacid, 1-dodecanoylaminoethane-2-sulfonic acid,1-tetradecanoylaminoethane-2-sulfonic acid,1-hexadecanoylaminoethane-2-sulfonic acid, and1-octadecanoylaminoethane-2-sulfonic acid; taurocholic acid andtaurodeoxycholic acid; bile acids and salts thereof, such as cholicacid, deoxycholic acid and sodium glycocholates; sodium caprate, sodiumlaurate, sodium oleate, sodium lauryl sulphate, sodium cetyl sulphate,sulfated castor oil, and sodium dioctylsulfosuccinate;cocamidopropylbetaine and laurylbetaine; fatty alcohols; cholesterols;glycerol mono- or -distearate, glycerol mono- or -dioleate, and glycerolmono- or -dipalmitate; and polyoxyethylene stearate.

Examples of sweetening agents include, without limitation, acesulfampotassium, isomalt, Magna Sweet, maltitol, mannitol, sorbitol,sucralose, xylitol, alitmae, neohesperidin dihydrochalcone, trehalose,tagatose, neotame, saccharin and salts thereof, stevioside, erythritol,isomaltulose, polydextrose, luo han guo, monatin, cyclamate,glycyrrhizin, osladine, sucrose, fructose, or glucose or combinationsthereof.

Non-limiting examples of flavoring agents include cherry, grape, orange,pink lemonade, raspberry, grape, lemon, orange, strawberry,tutti-frutti, tangerine, apple, watermelon, pineapple, banana, peach,kiwi, mango, mixed berry, raspberry lemonade, wild blackberry, blueraspberry, citrus, blueberry, lime, lemon lime, grapefruit, pomegranate,pear, or plum flavors, bubble gum, or combinations thereof.

Non-limiting examples of flavor enhancer agents include anhydrous citricacid, citric acid monohydrate, malic acid, tartaric acid, sodiumcitrate, potassium citratemonohydrate, potassium citrate anhydrous, orsodium potassium tartate or combinations thereof.

Non-limiting examples of coloring agents include, but are not limitedto, riboflavin, cochineal dye, carmine, blue No. 1 for food use, yellowNo. 4 aluminum lake for food use, yellow No. 5 aluminum lake for fooduse, red No. 3 aluminum lake for food use, red No. 106 for food use,iron sesquioxide, yellow iron sesquioxide, pharmaceutical dyes such asFD&C Blue No. 2, FD&C Blue No. 1, FD&C Green No. 3, FD&C Red No. 3, FD&CRed No. 4, FD&C Red No. 40, FD&C Yellow No. 6, FD&C Yellow No. 5, andthe like.

The pharmaceutical formulation in the stable sachet dosage formsdescribed herein optionally can also comprise other excipients, such asnon-reducing sugars, such as melezitose, planteose, and raffinose.Without intending to be bound by any particular theory, reducing sugarsmay react with BH4 under certain conditions. Other excipients useful incompositions described herein include phosphates, such as dicalciumphosphate.

The pharmaceutical formulation in the stable sachet dosage forms canoptionally include one or more other therapeutic agents suitable for thecondition to be treated. In one embodiment the therapeutic agent can beblended with the stable BH4 dihydrochloride powder. In anotherembodiment the therapeutic agent is contained in one chamber of a dualchamber sachet and the stable BH4 dihydrochloride dry blend powder is inthe second chamber. In one embodiment, the other therapeutic agents areselected from folates, including, but not limited to, folate precursors,folic acids and folate derivatives, e.g., folinic acid (leucovorin);vitamins, such as vitamin C (ascorbic acid), vitamin B2 (riboflavin),and vitamin B12; neurotransmitter precursors, such as L-dopa, carbidopa,and serotonin; 5-hydroxytryptophan; arginine; and combinations thereof.

Exemplary folates, including folate precursors, folic acids, and folatederivatives, are disclosed in U.S. Pat. Nos. 6,011,040 and 6,544,994(each of which is incorporated herein by reference in its entirety), andinclude folic acid (pteroylmonoglutamate), dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, one or more of the folylpolyglutamates, compounds in which thepyrazine ring of the pterin moiety of folic acid or of thefolylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, derivatives of all the preceding compounds in whichthe N-5 or N-10 position carries one carbon unit at various levels ofoxidation, pharmaceutically acceptable salts thereof, and combinationsof two or more thereof. Exemplary tetrahydrofolates include5-formyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methynyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5-formimino-(6S)-tetrahydrofolic acid, or (6S)-tetrahydrofolicacid, and pharmaceutically acceptable salts thereof. Exemplary saltsinclude sodium, potassium, calcium, and ammonium salts. Exemplaryrelative weight ratios of BH4 to folates to arginine can be in a rangefrom about 1:10:10 to about 10:1:1.

In a specific embodiment, the BH4 used in compositions described hereinis formulated as a dihydrochloride salt. Other salt forms of BH4possessing the desired physicochemical properties and biologicalactivity can also be used. For example, BH4 salts with inorganic ororganic acids are within the scope of the present disclosure.Nonlimiting examples of alternative BH4 salt forms include BH4 salts ofacetic acid, citric acid, oxalic acid, tartaric acid, fumaric acid, andmandelic acid. Carbonates or hydrogen carbonates are also possible.

Pharmaceutically acceptable salts can be formed with metals or amines,such as alkali and alkaline earth metals or organic amines.Pharmaceutically acceptable salts of compounds can also be prepared witha pharmaceutically acceptable cation. Suitable pharmaceuticallyacceptable cations are well known to those skilled in the art andinclude alkaline, alkaline earth, ammonium, and quaternary ammoniumcations. Non-limiting examples of metals used as cations are sodium,potassium, magnesium, ammonium, calcium, ferric, and the like.Non-limiting examples of suitable amines include isopropylamine,trimethylamine, histidine, N,N′-dibenzylethylenediamine, chloroprocaine,choline, diethanolamine, dicyclohexylamine, ethylenediamine,N-methylglucamine, and procaine.

Furthermore, the pharmaceutically acceptable salts include inorganic andorganic acid salts. Non-limiting examples of suitable salts includehydrochlorides, acetates, citrates, salicylates, nitrates, andphosphates. Other suitable pharmaceutically acceptable salts are wellknown to those skilled in the art and include those formed with, e.g.,acetic, citric, oxalic, tartaric or mandelic acid, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid; withorganic carboxylic, sulfonic, sulfo- or phosphor-acids or N-substitutedsulfamic acids, for example, acetic acid, phenylacetic acid, propionicacid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid,ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane 1,2-disulfonicacid, benzenesulfonic acid, 4-methylbenzenesulfonic acid,2-naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, or N-cyclohexylsulfamic acid(with the formation of cyclamates); with other acid organic compounds,such as ascorbic acid; or with amino acids, such as the 20 alpha aminoacids involved in the synthesis of proteins in nature, e.g., glutamicacid or aspartic acid.

In an embodiment, the pharmaceutical formulation in the stable sachetdosage forms contains about 5%, about 7.5%, about 10%, about 12.5%,about 15%, about 17.5%, about 20%, about 22.5%, about 25%, about 27.5%,about 30%, about 32.5%, about 35%, about 37.5%, about 40%, about 42.5%,about 45%, about 47.5%, about 50%, about 52.5%, or about 55% BH4dihydrochloride. In certain embodiments, the stable sachet dosage formscontain between about 10-50%, about 10-40%, about 10-30%, about 10-20%,about 20-50%, about 30-50%, about 40-50%, or about 50-55% BH4dihydrochloride.

In an embodiment, the pharmaceutical formulation in the stable sachetdosage forms comprise an amount of BH4 dihydrochloride of about 15% andbetween 75-80% mannitol, about 0-1% sucralose, about 1.5% flavoringagent, between 4.5-7% potassium citrate or potassium sodium tartrate,and about 1% ascorbic acid fine powder. In a specific embodiment, theflavoring agent is strawberry, orange on a sucrose substrate, orange ona mannitol substrate, or grape. In another specific embodiment, theblend also consists of about 5% potassium citrate.

In an embodiment, the pharmaceutical formulation in the stable sachetdosage forms comprise an amount of BH4 dihydrochloride of about 15.1%BH4 dihydrochloride, 75.3% mannitol, 1.5% orange flavor on a sucrosebase, 0.8% sucralose, 4.8% potassium citrate, and 0.8% ascorbic acidfine powder.

In an embodiment, the pharmaceutical formulation in the stable sachetdosage forms comprise an amount of BH4 dihydrochloride of about 15.3%BH4 dihydrochloride, 76.7% mannitol, 1.5% orange flavor on a mannitolbase, 0.8% sucralose, 4.8% potassium citrate, and 0.8% ascorbic acidfine powder.

In yet another embodiment, the pharmaceutical formulation in the stablesachet dosage forms additionally comprises an amount of5-hydroxytryptophan (5-HTP) in a range from about 0% to about 50% byweight of the formulation. In one embodiment, the 5-HTP is dry blendedwith the BH4 dihydrochloride. In another embodiment, the 5-HT iscontained in one chamber of a dual chamber sachet and the BH4dihydrochloride is contained in the second chamber. In narrowerembodiments, the formulation additionally comprises 5-HTP from about 5%to about 45%, or from about 10% to about 40%, or from about 20% to about40%, or from about 15% to about 35%, or from about 20% to about 30%, byweight of the formulation.

In yet another embodiment, the pharmaceutical formulations in the stablesachet dosage forms are blends that contain of approximately 32% drug,55% mannitol, and 1.6% to 2% sucralose. For example:

Formulation A Formulation B Formulation C Ingredient g/sachet % g/sachet% g/sachet % Sapropterin HCl 0.2 32 0.2 32 0.2 32 Mannitol 0.34 54.40.3388 54.2 0.3375 54.0 Sucralose 0.01 1.6 0.0113 1.8 0.0125 2.0Potassium Citrate 0.065 10.4 0.065 10.4 0.065 10.4 Ascorbic Acid 0.011.6 0.01 1.6 0.01 1.6 Total 0.625 100 0.625 100 0.625 100

In yet another embodiment, the pharmaceutical formulation in the stablesachet dosage contains approximately 32% drug, 54.1% mannitol, 1.9%sucralose micronized, 10.4% potassium citrate monohydrate, and 1.6%ascorbic acid fine powder.

In certain embodiments, the stable sachet dosage forms described hereincontain an amount of BH4 or a BH4-related compound (e.g.,(6R)-L-erythro-BH4 dihydrochloride) in a range from about 50 mg to about1300 mg (i.e., 1.3 g) per sachet, or from about 100 mg to about 1300 mgper sachet, or from about 200 mg to about 1300 mg per sachet, or fromabout 300 mg to about 1300 mg per sachet, or from about 400 mg to about1300 mg per sachet, or from about 500 mg to about 1300 mg per sachet, orfrom about 600 mg to about 1300 mg per sachet, or from about 700 mg toabout 1300 mg per sachet, or from about 800 mg to about 1300 mg persachet, or from about 900 mg to about 1300 mg per sachet, or from about1000 mg to about 1300 mg per sachet, or from about 50 mg to about 500 mgper sachet, or from about 100 mg to about 500 mg per sachet, or fromabout 200 mg to about 500 mg per sachet, or from about 300 mg to about500 mg per sachet, or from about 100 mg to about 700 mg per sachet, orfrom about 200 mg to about 700 mg per sachet, or from about 300 mg toabout 700 mg per sachet, or from about 400 mg to about 700 mg persachet. In specific embodiments, the stable sachet dosage forms containan initial amount of BH4 or a BH4-related compound of about 50 mg persachet, 100 mg per sachet, or about 150 mg per sachet, or about 160 mgper sachet, or about 200 mg per sachet, or about 250 mg per sachet, orabout 300 mg per sachet, or about 350 mg per sachet, or about 400 mg persachet, or about 450 mg per sachet, or about 500 mg per sachet, or about550 mg per sachet, or about 600 mg per sachet, or about 650 mg persachet, or about 700 mg per sachet, or about 750 mg per sachet, or about800 mg per sachet, or about 850 mg per sachet, or about 900 mg persachet, or about 950 mg per sachet, or about 1000 mg per sachet, orabout 1050 mg per sachet, or about 1100 mg per sachet, or about 1150 mgper sachet, or about 1200 mg per sachet, or about 1250 mg per sachet, orabout 1300 mg per sachet, or about 1350 mg per sachet. It will beapparent to one skilled in the art that the desired dosage for thetreatment, amelioration or prevention of a BH4-responsive disorder canaffect the amount of BH4 or a BH4-related compound in the sachet dosageforms.

In one embodiment, provided herein are sachet dosage forms that containrelatively large amounts of BH4 (e.g., (6R)-L-erythro-BH4dihydrochloride). A non-limiting example of such a dosage form includes500 mg of BH4, or 600 mg of BH4, or 700 mg of BH4, or 800 mg of BH4, or900 mg of BH4, or 1000 mg of BH4, or 1100 mg of BH4, or 1200 mg of BH4,or 1300 mg of BH4. Potential advantages of relatively large amounts ofBH4 available in a dosage form include ease of providing thetherapeutic, ease of oral administration, and patient compliance.

It is particularly unexpected that weights of about 600 mg or more, forexample, of a dry blend powder have flow properties that are amenable toautomated sachet filling. Accordingly, it is unexpected that stablesachet dosage forms described herein contain, for example, 600 mg ormore of a BH4 or BH4-related compound dry blend powder.

In an embodiment, the stable sachet dosage forms described herein areuseful for treating BH4 deficiency or a condition or disorder associatedwith BH4 deficiency (e.g., hyperphenylalaninemia due to BH4 deficiency).In a specific embodiment, the stable sachet dosage is used reduce bloodphenylalanine levels in patients with hyperphenylalaninemia due totetrahydrobiopterin-responsive phenylketonuria. In another specificembodiment, the stable sachet dosage is used reduce blood phenylalaninelevels in patients with hyperphenylalaninemia due totetrahydrobiopterin-responsive phenylketonuria in conjunction with aphenylalanine restricted diet.

In one embodiment, the stable sachet dosage forms useful for treatingBH4 deficiency or a condition associated therewith comprise amounts of(1) BH4 or a BH4-related compound (e.g., (6R)-L-erythro-BH4dihydrochloride) from about 30% to about 60%, or from about 40% to about60%; (2) mannitol from about 20% to about 50%, or from about 30% toabout 50%; (3) crospovidone from about 2% to about 8%, or from about 3%to about 6%; (4) stearyl fumaric acid or a salt form of stearyl fumarate(e.g., sodium stearyl fumarate) from about 1% to about 4%, or from about1% to about 3%; (5) ascorbic acid from about 1% to about 20%, or fromabout 1% to about 10%; and (6) silicon dioxide (e.g., colloidal silicondioxide) from about 0.2% to about 4%, or from about 0.2% to about 2%, byweight of the formulation. In a related embodiment, the stable sachetuseful for treating BH4 deficiency or a condition associated therewithcomprise an initial amount of BH4 or a BH4-related compound (e.g.,(6R)-L-erythro-BH4 dihydrochloride) in a range from about 50 mg to about1300 mg per sachet, including but not limited to about 50 mg per sachet,100 mg per sachet, or about 150 mg per sachet, or about 160 mg persachet, or about 200 mg per sachet, or about 250 mg per sachet, or about300 mg per sachet, or about 350 mg per sachet, or about 400 mg persachet, or about 450 mg per sachet, or about 500 mg per sachet, or about550 mg per sachet, or about 600 mg per sachet, or about 650 mg persachet, or about 700 mg per sachet, or about 750 mg per sachet, or about800 mg per sachet, or about 850 mg per sachet, or about 900 mg persachet, or about 950 mg per sachet, or about 1000 mg per sachet, orabout 1050 mg per sachet, or about 1100 mg per sachet, or about 1150 mgper sachet, or about 1200 mg per sachet, or about 1250 mg per sachet, orabout 1300 mg per sachet, or about 1350 mg per sachet.

In a further embodiment, the stable sachet dosage forms described hereinare useful for treating sickle cell disease (SCD), peripheral arterialdisease (PAD), chronic kidney disease (CKD), or hypertension. In oneembodiment, the stable sachet dosage forms useful for treating SCD, PAD,CKD, or hypertension comprise initial amounts of (1) BH4 or aBH4-related compound (e.g., (6R)-L-erythro-BH4 dihydrochloride) fromabout 30% to about 60%, or from about 40% to about 60%, or from about40% to about 50%; (2) ascorbic acid from about 30% to about 60%, or fromabout 40% to about 60%, or from about 40% to about 50%; (3) crospovidonefrom about 2% to about 8%, or from about 3% to about 6%; (4) stearylfumaric acid or a salt form of stearyl fumarate (e.g., sodium stearylfumarate) from about 1% to about 4%, or from about 1% to about 3%; (5)silicon dioxide (e.g., colloidal silicon dioxide) from about 0.2% toabout 4%, or from about 0.2% to about 2%; and (6)5-methyltetrahydrofolate (5-MTHF) or a salt form thereof from about 0%to about 2%, or from about 0.01% to about 1%, or from about 0.01% toabout 0.5%, by weight of the formulation. The 5-MTHF can be apharmaceutically acceptable salt of 5-MTHF, e.g., the calcium salt of5-MTHF. In a related embodiment, the stable sachets useful for treatingSCD, PAD, CKD, or hypertension comprise initial amounts of BH4 or aBH4-related compound (e.g., (6R)-L-erythro-BH4 dihydrochloride) andascorbic acid in a weight ratio of about 10:1, about 9:1, about 8:1,about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2.5:1,about 2:1, about 1.5:1, about 1:1, about 1:1.5, or about 1:2. In anotherrelated embodiment, the stable sachets useful for treating SCD, PAD,CKD, or hypertension comprise an initial amount of BH4 or a BH4-relatedcompound (e.g., (6R)-L-erythro-BH4 dihydrochloride) in a range fromabout 50 mg to about 1300 mg per sachet, including but not limited about50 mg per sachet, 100 mg per sachet, or about 150 mg per sachet, orabout 160 mg per sachet, or about 200 mg per sachet, or about 250 mg persachet, or about 300 mg per sachet, or about 350 mg per sachet, or about400 mg per sachet, or about 450 mg per sachet, or about 500 mg persachet, or about 550 mg per sachet, or about 600 mg per sachet, or about650 mg per sachet, or about 700 mg per sachet, or about 750 mg persachet, or about 800 mg per sachet, or about 850 mg per sachet, or about900 mg per sachet, or about 950 mg per sachet, or about 1000 mg persachet, or about 1050 mg per sachet, or about 1100 mg per sachet, orabout 1150 mg per sachet, or about 1200 mg per sachet, or about 1250 mgper sachet, or about 1300 mg per sachet, or about 1350 mg per sachet.

In a particular embodiment of a stable sachet dosage form useful fortreating SCD, PAD, CKD, or hypertension, the pharmaceutical formulationof the dosage form comprises the following ingredients, in terms ofweight % of the formulation and mg per capsule: (1) 46.75%(6R)-L-erythro-BH4 dihydrochloride (250.0 mg); (2) 46.75% ascorbic acid(250.0 mg); (3) 3.98% or 3.96% crospovidone (21.3 mg or 21.2 mg); (4)1.75% sodium stearyl fumarate (9.4 mg); (5) 0.75% colloidal silicondioxide (4.0 mg); and (6) 0.02% or 0.04% 5-methyltetrahydrofolate,calcium salt (0.1 mg or 0.2 mg).

In another particular embodiment, the stable sachet dosage form can beused for treating or ameliorating autism. BH4 has been shown to beeffective in treating autism in humans (see, e.g., Fernell et al., Dev.Med. & Child Neurology, 39: 313-318 (1997); Frye et al.Neurotherapeutics, 7: 241-249; Danfors et al., J. ClinicalPsychopharmacology, 25(5): 485-489). Fernell shows that children treatedwith BH4 for 4, 8, and 12 weeks had significant increases in their totalscores on the Parental Satisfaction Survey (“PSS”). Fernell, Table II,page 316. Frye reviews research using BH4 to treat autism and concludesthat BH4 represents a novel therapy for autism spectrum disorder.Danfors shows that children treated with BH4 had significant improvementof their social interaction scores after 6 months of treatment and founda high positive correlation between response of the social interactionscore and IQ.

In one embodiment, the BH4 or BH4-related compound can be used fortreating or ameliorating autism in children. In one embodiment, the BH4or BH4-related compound can be used for treating or ameliorating autismin adults. In a particular embodiment, the BH4 or BH4-related compoundcan be administered in conjunction with a second pharmaceuticalcomposition to treat or ameliorate the symptoms of autism. In aparticular embodiment, the second pharmaceutical compound for thecombination treatment can be selected from groups consisting ofstimulants, antidepressants, antianxiety medications, non-stimulant ADHDmedications, antipsychotics, mood stabilizers, or Alzheimer'smedications.

Sachet dosage forms can be manufactured by dry blending the componentsof the sachet and sealing them in a single or dual chamber sachet. Thesachet can be flushed with an inert gas and hermetically sealed. Thesachet can also contain a desiccant. The sachet can be made out of Mylarfoil. Further, the sachet can then be filled with the compoundedmaterial and, optionally, the capsule can be stored in a sealedcontainer, e.g., a Mylar foil package. The Mylar foil package maycontain a desiccant and may be flushed with inert gas and may behermetically sealed. The compounded pharmaceutical components or powderblends can be uniformly filled into sachets using an automatedsachet-filling equipment or can be manually filled into sachets, as isknown in the art.

In an embodiment, the stable sachet dosage forms, including thepharmaceutical formulations contained therein, are made by a processthat does not include adding an aqueous liquid. The dry blendmanufacturing process comprises a dry process of mixing and blending aninitial amount of the active ingredient, BH4 or a BH4-related compound(e.g., (6R)-L-erythro-BH4 dihydrochloride), with an initial amount of anexcipient that optionally has been screened with an appropriate meshscreen, optionally mixing and blending the resulting ingredients with aninitial amount of a second excipient that optionally has been screened,optionally doing the same with additional excipients, and filling theresulting dry blend of ingredients into capsules.

In another embodiment, the methods involve the step of informing thepatient that absorption of tetrahydrobiopterin is increased when it isingested with food compared to when ingested without food. In someembodiments, the patient is informed that ingestion shortly following ameal, for example, a high-fat, high-calorie meal, results in an increasein any one, two, three or all of the following parameters: mean plasmaconcentration, Cmax, AUC, AUC(0-t) and/or AUC(inf). In otherembodiments, the patient is informed that administration of BH4 with ahigh-fat meal increases Cmax and AUC compared to administration of BH4without food (in a fasting condition). In some embodiments, the relativeincrease can be at least 20% or 30% or more.

In another embodiment, such methods involve administering BH4, whetherswallowed as a solid or semisolid dosage form, or dissolved in a liquid,with food, e.g., a high-fat food or a high-fat and/or high-calorie meal.In another embodiment, BH4, whether swallowed or dissolved, isadministered at a specified time including but not limited to morning,day, night, same time of the day, with food, e.g., a high-fat food or ahigh-fat and/or high-calorie meal, one or more times a day. In anotherembodiment, BH4 is ingested once daily as a solid dosage form just aftermeals. In another embodiment the solid dosage form is a formulatedtablet or capsule. In other embodiments, BH4 is ingested withinapproximately 0 to 30 minutes, or 5 to 20 minutes, of eating a meal.

The BH4 and the food may be ingested at approximately the same time, orthe BH4 may be ingested before or after the food. The period of timebetween consuming the food and taking BH4, either swallowed ordissolved, may be at least 5 minutes. For example, BH4 may beadministered 60 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes,10 minutes, or 5 minutes before or after a meal.

In another embodiment, to maximize oral bioavailability of BH4 at eachadministration, BH4 should be taken with food, e.g., a high fat food ora high fat and/or high calorie meal. Alternatively, to maximizeconsistency of oral bioavailability between administrations, BH4 shouldbe taken on an empty stomach (e.g., 1 hour before or 2 hours after ameal).

The container or packaging containing BH4 sachet dosage forms isselected, inter alia, to minimize or prevent moisture penetration intoand contamination of the formulation in the dosage form and therebyenhance the stability of the formulation. In an embodiment, BH4 sachetdosage forms are disposed in a sealed container or packaging. Examplesof sealed containers include Mylar foil pouches. Additional non-limitingexamples of sealed containers or packaging include polyethylene bottles,e.g., high density polyethylene bottles closed with a heat-inductionseal, glass bottles, tubes, hermetically sealed foil packaging,thermally sealed polyethylene bags, laminated foil pouches, and blisterpacks. In one embodiment, a desiccant is disposed in the container orpackaging containing the BH4 sachet dosage forms, or is incorporated inthe closure, stopper or cap of the container or packaging. In oneembodiment the container is flushed with inert gas prior to beinghermetically sealed.

In another embodiment, at least about 90% of the initial amount of theBH4 or BH4-related compound remains, after the sachet dosage form isstored at about 40° C. and about 75% RH for a period of about threemonths. In another embodiment, at least about 91%, or at least about92%, or at least about 93%, or at least about 94%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99% of the initial amount of BH4 or BH4-relatedcompound remains after the sachet dosage form is stored at about 40° C.and about 75% RH for a period of about three months.

In another embodiment, at least about 85% of the initial amount of theBH4 or BH4-related compound dissolves within about 15 minutes, after thesachet dosage form is stored at about 40° C. and about 75% RH for aperiod of about three months. In another embodiment, at least about 86%,or at least 87%, or at least 88%, or at least 89%, or at least 90%, orat least 91%, or at least 92%, or at least 93%, or at least 94%, or atleast 95%, or at least 96%, or at least 97%, or at least 98%, or atleast 99% of the initial amount of the BH4 or BH4-related compounddissolves within about 15 minutes, after the sachet dosage form isstored at about 40° C. and about 75% RH for a period of about threemonths.

In another embodiment, at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% of the initial amount of the BH4or BH4-related compound remains and at least about 85%, or at leastabout 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99% of the initial amount of the BH4 orBH4-related compound dissolves within about 15 minutes, after the sachetdosage form is stored at about 40° C. and about 75% RH for a period ofabout three months.

In another embodiment, at least about 85%, or at least about 86%, or atleast 87%, or at least 88%, or at least 89%, or at least 90%, or atleast 91%, or at least 92%, or at least 93%, or at least 94%, or atleast 95%, or at least 96%, or at least 97%, or at least 98%, or atleast 99% of the initial amount of the BH4 or BH4-related compounddissolves within about 1 minute, or within about 2 minutes, or withinabout 3 minutes, or within about 4 minutes, or within about 5 minutes,or within about 6 minutes, or within about 7 minutes, or within about 8minutes, or within about 9 minutes, or within about 10 minutes withgentle agitation, after the sachet dosage form is stored at about 40° C.and about 75% RH for a period of about three months. In certainembodiments the gentle agitation is stirring or shaking.

In another embodiment, at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% of the initial amount of the BH4or BH4-related compound remains and at least about 85%, or at leastabout 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99% of the initial amount of the BH4 orBH4-related compound dissolves within about 1 minute, or within about 2minutes, or within about 3 minutes, or within about 4 minutes, or withinabout 5 minutes, or within about 6 minutes, or within about 7 minutes,or within about 8 minutes, or within about 9 minutes, or within about 10minutes with gentle agitation, after the sachet dosage form is stored atabout 40° C. and about 75% RH for a period of about three months. Incertain embodiments the gentle agitation is stirring or shaking.

Therapeutic Methods Using Stable Dosage Forms

It will be appreciated that therapeutic methods applicable to the stabledosage forms described herein include the fields of human medicine andveterinary medicine. Thus, the subject to be treated can be a mammal,e.g., human or other animal.

The stable dosage forms described herein can be used for treating,ameliorating or preventing, e.g., conditions associated with elevatedphenylalanine levels or decreased tyrosine or tryptophan levels. Suchconditions may be caused, for example, by reduced phenylalaninehydroxylase activity or reduced tyrosine hydroxylase or tryptophanhydroxylase activity. Conditions associated with elevated phenylalaninelevels include, e.g., phenylketonuria, both mild and classic, andhyperphenylalanemia (also written as hyperphenylalaninemia), andexemplary patient populations include the patient subgroups describedherein as well as any other patients exhibiting phenylalanine levelsabove normal. Conditions associated with reduced tyrosine or tryptophanlevels include, e.g., neurotransmitter deficiency, neurological andpsychiatric disorders such as Parkinson's, dystonia, spinocerebellardegeneration, pain, fatigue, depression, other affective disorders, andschizophrenia. Non-limiting examples of other BH4-responsive conditionsthat can be treated, ameliorated or prevented by use of the BH4 stablecapsule dosage forms described herein include BH4 deficiency, sicklecell anemia, hypertension and autism.

Genetic disorders resulting in hyperphenylalaninemia (HPA) involvedeficiency in the enzyme phenylalanine hydroxylase (PAH), which causesphenylketonuria (PKU), and deficiency in the essential PAH cofactor,tetrahydrobiopterin (BH4), which causes disorders known as BH4deficiency. BH4 is an essential cofactor for several enzymes, includingPAH, tyrosine hydroxylase, tryptophan hydroxylase, all three nitricoxide synthetase (NOS) isoforms, and glyceryl-ether monooxygenase. BH4deficiency can result from defects in the genes encoding the enzymesinvolved in BH4 synthesis (GTP cyclohydrolase 1 (GTP-CH 1),6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase(SR)) or BH4 regeneration (dihydropteridine reductase (DHPR) andpterin-4-carbinolamine dehydratase (PCD, also called 4a-carbinolaminedehydratase)). In addition to elevated blood phenylalanine levels (HPA),patients with defects in BH4 metabolism can suffer progressiveneurologic deterioration due to decreased production of theneurotransmitters dopamine, epinephrine, norepinephrine and serotonin.

The BH4 stable dosage forms described herein can be used to treat,ameliorate or prevent BH4 deficiency associated with deficiency in orreduced activity of any one or any combination of the enzymes involvedin BH4 synthesis or recycling—GTP-CH 1, PTPS, SR, DHPR and PCD. Forexample, the BH4 stable dosage forms can be used to treat, ameliorate orprevent hyperphenylalanemia due to BH4 deficiency and associated withdeficiency in or reduced activity of any one or any combination of theaforementioned enzymes. The BH4 stable dosage forms can also be used totreat, ameliorate or prevent hyperphenylalanemia due to phenylketonuria.In an embodiment, the BH4 stable dosage forms can be administered inconjunction with a low phenylalanine diet; with the neurotransmitterprecursors L-dopa, carbidopa and/or serotonin; with folinic acid(leucovorin) or a salt form of folinate (e.g., calcium folinate), e.g.,in the case of DHPR deficiency; or with any combinations thereof.

Suitable subjects for treatment with the stable dosage forms describedherein include subjects with an elevated plasma Phe concentration in theabsence of the therapeutic, e.g., greater than about 180 uM, greaterthan about 200 uM, greater than about 300 uM, greater than about 400 uM,greater than about 500 uM, greater than about 600 uM, greater than about800 uM, greater than about 1,000 uM, greater than about 1,200 uM,greater than about 1,400 uM, greater than about 1,600 uM, or greaterthan about 1,800 uM. Mild PKU is generally classified as plasma Pheconcentrations of up to about 600 uM, moderate PKU as plasma Pheconcentrations of between about 600 uM and about 1,200 uM, and classicor severe PKU as plasma Phe concentrations that are greater than about1,200 uM. Treatment with the dosage form alone or with aprotein-restricted diet can decrease the plasma phenylalanineconcentration of a subject to less than about 600 uM, less than about500 uM, less than about 400 uM, less than about 350 uM, less than about300 uM, less than about 250 uM, less than about 200 uM, less than about150 uM, or less than about 100 uM.

Other suitable subjects include subjects diagnosed as having a reducedphenylalanine hydroxylase (PAH) activity. Reduced PAH activity mayresult from a mutation in the PAH enzyme, for example, a mutation in thecatalytic domain of PAH or one or more mutations selected from the groupconsisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A,P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G,A395P, P407S, and Y414C.

Further, other suitable subjects can include pregnant females, femalesof child-bearing age who are contemplating pregnancy, infants between 0and 3 years of age (e.g., 0-2, 0-1.5 or 0-1 year old), and subjectsdiagnosed as being unresponsive within 24 hours to a single-dose BH4loading test or a multiple dose loading test, such as a 4-dose or 7-dayloading test. Exemplary patient populations and exemplary BH4 loadingtests are described in WO 2005/049000, which is incorporated herein byreference in its entirety.

U.S. Pat. Nos. 4,752,573; 4,758,571; 4,774,244; 4,920,122; 5,753,656;5,922,713; 5,874,433; 5,945,452; 6,274,581; 6,410,535; 6,441,038; and6,544,994; and U.S. Application Publication Nos. US 2002/0187958; US2002/0106645; US 2002/0076782; and US 2003/0032616 (each incorporatedherein by reference in its entirety), each describe methods ofadministering BH4 compositions for non-PKU treatments. Each of thesepatent documents is incorporated herein by reference as teaching methodsof administering BH4 compositions known to those of skill in the art,which can be adapted for the therapeutic method described herein, aswell as teaching related conditions that can also be treated by applyingthe present disclosure.

While individual needs may vary, determination of the optimal range ofthe effective amount of the active ingredient and the optimaladministration regimen is within the skill of an ordinary artisan.Exemplary dosages of BH4 or a BH4-related compound can range from about1 to about 30 mg/kg body weight per day, which may amount to about 5 (1mg/kg×5 kg body weight) to 3,000 mg/day (30 mg/kg×100 kg body weight).Dosages of BH4 or a BH4-related compound can also range from about 2 toabout 20 mg/kg/day. One embodiment is directed to daily administrationof a BH4 capsule dosage form for a continuous period of time. Forhyperphenylalanemia, however, it may be desirable to cease the BH4therapy when the symptoms of Phe levels are reduced to below a certainthreshold level. Of course, the BH4 therapy can be reinitiated in theevent that Phe levels rise again. Appropriate dosages can be ascertainedthrough use of established assays for determining blood levels of Phe inconjunction with relevant dose response data.

In an embodiment, the method of the present disclosure provides to apatient a daily dose of between about 10 mg/kg and about 20 mg/kg of BH4or a BH4-related compound. Of course, one skilled in the art can adjust(i.e., increase or decrease) the dose depending on the efficacy achievedby the administration. The daily dose can be administered in a singledose or alternatively can be administered in multiple doses atconveniently spaced intervals. In exemplary embodiments, the mg/kg dailydose of BH4 or a BH4-related compound can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70 or more mg/kg body weightper day.

The BH4 stable dosage forms described herein can also be used to treatsubjects suffering from conditions or disorders that would benefit fromenhancement of nitric oxide synthase (NOS) activity and subjectssuffering from vascular diseases, ischemic or inflammatory diseases, orinsulin resistance. Unless expressly indicated otherwise, the term“nitric oxide synthase” or “NOS” refers to all the isoforms of NOS,i.e., endothelial nitric oxide synthase (eNOS), neuronal nitric oxidesynthase (nNOS), and inducible nitric oxide synthase (iNOS). The BH4treatment can, e.g., alleviate a deficiency in NOS activity or can,e.g., provide an increase in NOS activity over normal levels.

Nitric oxide is constitutively produced by vascular endothelial cells,where it plays a key physiological role in the regulation of bloodpressure and vascular tone. A deficiency in nitric oxide bioactivity maybe involved in the pathogenesis of vascular dysfunctions, includingcoronary artery disease, atherosclerosis of any arteries, includingcoronary, carotid, cerebral and peripheral vascular arteries,ischemia-reperfusion injury, hypertension, diabetes, diabeticvasculopathy, cardiovascular disease, peripheral vascular disease, andneurodegenerative conditions stemming from ischemia and/or inflammation,such as stroke. Such pathogenesis includes damaged endothelium,insufficient oxygen flow to organs and tissues, elevated systemicvascular resistance (high blood pressure), vascular smooth muscleproliferation, progression of vascular stenosis (narrowing) andinflammation. In an embodiment, the BH4 capsule dosage forms describedherein can be used to treat any of the aforementioned conditions.

Enhancement of nitric oxide synthase activity may also result inreduction of superoxide levels, increased insulin sensitivity, andreduction in vascular dysfunction associated with insulin resistance, asdescribed in U.S. Pat. No. 6,410,535, which is incorporated herein byreference in its entirety. Thus, the BH4 dosage forms described hereincan also be used to treat diabetes (type I and type II),hyperinsulinemia, and insulin resistance. Diseases having vasculardysfunction associated with insulin resistance include those caused byinsulin resistance or aggravated by insulin resistance, or those forwhich cure is retarded by insulin resistance, such as hypertension,hyperlipidemia, arteriosclerosis, coronary vasoconstrictive angina,effort angina, cerebrovascular constrictive lesion, cerebrovascularinsufficiency, cerebral vasospasm, peripheral circulation disorder,coronary arteriorestenosis following percutaneous transluminal coronaryangioplasty (PTCA) or coronary artery bypass grafting (CABG), obesity,insulin-independent diabetes, hyperinsulinemia, lipid metabolismabnormality, coronary arteriosclerotic heart diseases, and likeconditions that are associated with insulin resistance.

Moreover, the BH4 dosage forms described herein can be used to treatdrug-induced renal injury. The BH4 treatment can recover the functionsof endothelial cells and normalize the functions of NOS to provide renalprotective effects. Clinical states of drug-induced renal injury aretypically classified into acute renal failure and chronic renal failure,as described in U.S. Pat. No. 6,288,067, which is incorporated herein byreference in its entirety.

When administered to patients susceptible to or suffering from any ofthe aforementioned disorders, BH4 or a BH4-related compound can treat orprevent these disorders by activating the functions of NOS, increasingNO production, and suppressing the production of active oxygen species(e.g., superoxide), to improve disorders of vascular endothelial cells.

A subject suffering from a deficiency in nitric oxide synthase (NOS)activity may benefit from co-treatment with BH4 or a BH4-relatedcompounds, and folic acids, folates, folate precursors, or folatederivatives. Accordingly, in one embodiment, provided herein is use ofstable capsule dosage forms comprising BH4 or a BH4-related compoundsand a folic acid, folate, folate precursor, or folate derivative fortreating, ameliorating or preventing cardiovascular or neurologicaldisorders by modulation of the activity of a nitric oxide synthase(NOS). Modulation of the activity of an NOS can, e.g., reduce superoxide(O₂′) production, enhance nitric oxide (NO) synthesis, and influence NOlevels. U.S. Pat. Nos. 6,995,158, 6,544,994; and 6,011,040, each ofwhich is incorporated herein by reference in its entirety, describe theuse of BH4 and a folic acid or folate compound to modulate NOS activity.

Non-limiting examples of folic acids, folates, folate precursors, andfolate derivatives that can be used in the present disclosure includefolic acid (pteroylmonoglutamate), dihydrofolic acid, dihydrofolates,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, tetrahydrofolates, folylpolyglutamates, compounds in which thepyrazine ring of the pterin moiety of folic acid or of thefolylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, derivatives of all the preceding compounds in whichthe N-5 or N-10 position carries one carbon unit at various levels ofoxidation, pharmaceutically acceptable salts of all the precedingcompounds, and combinations thereof. In an embodiment, theBH4-containing formulation comprises an initial amount of a folic acid,a folate, a folate precursor or a folate derivative (e.g., folic acid or5-methyltetrahydrofolate) in a range from about 0.0005% to about 5%, orto about 3%, or to about 1%, or to about 0.5%, or to about 0.1%, or toabout 0.05% by weight of the formulation.

Exemplary tetrahydrofolic acids and tetrahydrofolates include5-formyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5-formimino-(6S)-tetrahydrofolic acid, (6S)-tetrahydrofolic acid,and pharmaceutically acceptable salts thereof. Exemplary salts of allthe folic acid and folate compounds described herein include sodium,potassium, calcium, and ammonium salts thereof.

The pharmaceutical formulation comprising BH4 or a BH4-related compoundand a folic acid, folate, folate precursor, or folate derivative, canfurther comprise one or more other active or adjuvant substances. In oneembodiment, the one or more other active or adjuvant substances includearginine (e.g., L-arginine). L-arginine is a precursor of endogenousnitric oxide (NO), a potent vasodilator acting via the intracellularsecond messenger cGMP. L-arginine induces peripheral vasodilation andinhibits platelet aggregation due to increased NO production. A lack ofNO production has been linked to pathological conditions, includingcardiovascular disorders (e.g., atherosclerosis), septic shock,inflammation and infection, and brain damage in stroke and neurologicaldisorders. In an embodiment, the formulation comprises BH4 or aBH4-related compound, a folic acid or a folate (which can also be afolate precursor or a folate derivative), and arginine each in a unitdosage from about 0.1 mg to about 250 mg. In another embodiment, theweight ratio of BH4 or a BH4-related compound to a folic acid or afolate to arginine ranges from about 1:10:10 to about 10:1:1.

In another embodiment, the pharmaceutical formulation comprising BH4 ora BH4-related compound can be used to treat or ameliorate autism. In aparticular embodiment, the BH4 or BH4-related compound is BH4dihydrochloride. In a particular embodiment, BH4 dihydrochloride can beadministered to children suffering from autism. In particularembodiment, the child is about 0-18 years old, 1-18 years old, about2-18 years old, about 3-18 years old, about 3-17 years old, about 3-16years old, about 3-15 years old, about 3-14 years old, about 3-13 yearsold, about 3-12 years old, about 3-11 years old, about 3-10 years old,about 3-9 years old, about 3-8 years old, about 3-7 years old, or about3-6 years old. In a particular embodiment, the is administered to asubject during childhood and adulthood. In a particular embodiment, theformulation can be administered at a dosage of about 5-50 mg/kg/day. Ina particular embodiment, the formulation can be administered at a dosageof about 5 mg/kg/day, or about 7.5 mg/kg/day, or about 10 mg/kg/day, orabout 10.5 mg/kg/day, or about 11 mg/kg/day, or about 11.5 mg/kg/day, orabout 12 mg/kg/day, or about 12.5 mg/kg/day, or about 13 mg/kg/day, orabout 13.5 mg/kg/day, or about 14 mg/kg/day, or about 14.5 mg/kg/day, orabout 15 mg/kg/day, or about 15.5 mg/kg/day, or about 16 mg/kg/day, orabout 16.5 mg/kg/day, or about 17 mg/kg/day, or about 17.5 mg/kg/day, orabout 18 mg/kg/day, or about 18.5 mg/kg/day, or about 19 mg/kg/day, orabout 19.5 mg/kg/day, or about 20 mg/kg/day. In another embodiment, thepharmaceutical formulation comprising BH4 or a BH4-related compound canbe used to treat or ameliorate autism at a dosage of 25 mg/kg/day ormore.

It is understood that the suitable dose will depend upon variousfactors, such as the age, health, weight, and diet of the recipient, thekind of concurrent treatment, if any, the frequency of treatment, andthe nature of the effect desired (e.g., the amount of decrease in plasmaPhe concentration desired). The frequency of dosing also depends on thepharmacodynamic effects of a BH4 dosage form. For example, if thepharmacological effects of a single dose of a BH4 capsule dosage formlast for about 24 hours, the BH4 capsule dosage form can be administeredonce daily. Alternatively, a BH4 dosage form can be administered two ormore times a day at appropriate time intervals in cases where multipledaily administrations to a particular subject are deemed to be moreeffective for treating, ameliorating or preventing a particularBH4-responsive condition. One or more BH4 doses can be administered in aparticular time of administration. For example, 1 BH4 dosage can betaken orally per day. As another example, 2 BH4 doses can be takenorally per day—e.g., 2 capsules or sachets once daily or 1 capsule orsachet twice daily. As yet another example, 3 BH4 doses can be takenorally per day—e.g., 3 capsules or sachets once daily, 2 capsules orsachets and 1 capsule or sachet at two different times, or 1 capsule orsachet thrice daily. As a further example, 4 BH4 doses can be takenorally per day—e.g., 4 capsules or sachets once daily, 2 capsules orsachets twice daily, 2 capsules or sachets and 2×1 capsule or sachets atthree different times, or 1 capsule or sachet four times daily. As stillanother example, 6 BH4 doses can be taken orally per day—e.g., 6capsules or sachets once daily, 3 capsules or sachets twice daily, 4capsules or sachets and 2 capsules or sachets at two different times, or2 capsules or sachets thrice daily. The desired dosage of the activeingredient, BH4 or a BH4-related compound, in a stable dosage form andthe frequency of administration of the dosage form can be tailored tothe individual subject and the particular BH4-responsive condition(s)being treated, as is understood and determinable by one of skill in theart, without undue experimentation. For example, a standard dose can beadjusted according to the facts and circumstances of a particularpatient, e.g., reduction of the dose if the patient has a low bodyweight.

In addition, the frequency of BH4 dosing will depend on thepharmacokinetic parameters of the active ingredient and the route/modeof administration. The optimal features of a BH4 capsule or sachetdosage form can be determined by one of skill in the art depending onvarious factors, such as the route of administration and the desireddosage. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., pp.1435-1712, Mack Publishing Co. (Easton, Pa., 1990), which isincorporated herein by reference. Depending on the route/mode ofadministration, a suitable dose can be calculated according to bodyweight, body surface areas or organ size. Further refinement of thecalculations for determining the appropriate treatment dose is routinelymade by those of ordinary skill in the art without undueexperimentation, including in light of the dosage information and assaysdisclosed herein as well as the pharmacokinetic data observed in animalsor human clinical trials.

The final dosage regimen will be determined by the attending physician,considering factors that may affect the action of the drug, e.g., thedrug's specific activity, the severity of the damage, the responsivenessof the patient, the age, condition, body weight, sex and diet of thepatient, the severity of any infection, the time and frequency ofadministration, and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions.

Combination Therapy

In one embodiment, the BH4 dosage forms described herein can be used incombination with one or more other therapeutic agents. In suchcombination therapy, administration of the BH4 dosage forms can beconcurrent with or can precede or follow the administration of a secondtherapeutic agent, e.g., by intervals ranging from minutes to hours. BH4or a BH4-related compound and the other therapeutic agent(s) can exerttheir pharmacological effects at overlapping or non-overlapping timeperiods. The disclosure encompasses the use of one or more additionaltherapeutic agents in the preparation of a pharmaceutical compositionfor use with BH4 or a BH4-related compound. The composition containingthe additional therapeutic agent(s) can also contain BH4 or aBH4-related compound or can be distinct from the capsule formulationcontaining BH4 or a BH4-related compound.

Tetrahydrobiopterin therapy can be combined with dietary proteinrestriction to effect a therapeutic outcome in patients with variousforms of hyperphenylalaninemia (HPA). For example, a subject can beadministered a BH4 dosage form and a low phenylalanine medical proteincomposition in a combined amount effective to produce the desiredtherapeutic outcome, i.e., a lowering of plasma Phe concentration and/orthe ability to tolerate greater amounts of Phe/protein intake withoutproducing a concomitant increase in plasma Phe concentrations. Theprocess can involve administering the BH4 dosage form and the dietaryprotein therapeutic composition at about the same time. This can beachieved by administering a single dosage form, or the dietary protein(supplement or normal protein meal) can be taken at about the same timeas the BH4 dosage form.

Alternatively, the BH4 treatment can precede or follow the dietaryprotein therapy by intervals ranging from minutes to hours. The proteincomposition and the BH4 dosage form can be administered separately suchthat the BH4 will still be able to exert an advantageous effect on thepatient. For example, the BH4 dosage form can be administered withinabout 1-6 hours (before or after) of the dietary protein intake. In amore specific example, the delay time between BH4 and dietary proteinadministrations can be about one hour. In one embodiment, the BH4therapy is a continuous therapy wherein a daily dose of BH4 isadministered to the patient continuously. In another embodiment, e.g.,in pregnant women having only the milder forms of PKU or HPA, the BH4therapy is only continued for as long as the woman is pregnant and/orbreast feeding.

In another embodiment, the therapeutic method provided herein is acombination therapy that administers BH4 or a BH4-related compound and acomposition that specifically targets one or more of the symptoms ofHPA. For example, the deficit in tyrosine associated with HPA results ina deficiency in the neurotransmitters dopamine and serotonin. Thus, aBH4 stable dosage form can be administered in combination with theneurotransmitter precursors L-dopa, carbidopa and/or5-hydroxytryptophan, with or without dietary protein therapy, to correctdefects that result from decreased amounts of tyrosine in the body.

In another embodiment, the therapeutic method provided herein is acombination therapy that administers BH4 or a BH4-related compound and acomposition that specifically targets one or more of the symptoms ofautism. For example, medications for depression, anxiety, ADD/ADHD, moodstabilization, and Alzheimer's disease can be used in combinationtherapy with BH4 or a BH4-related compounds to treat or ameliorate thesymptoms of autism. More specifically, for example stimulants (i.e.,ADD/ADHD treatment) administered in conjunction with the BH4 orBH4-related compound to treat and ameliorate autism may include but arenot limited to amphetamines, methylphenidate, pemoline,dextroamphetamines, dexmethylphenidate, methylphenidate, andcombinations thereof; non-stimulants (i.e., ADD/ADHD treatment)administered in conjunction with the BH4 or BH4-related compound totreat and ameliorate autism may include but is not limited toatomoxetine; antidepressants and anti-anxiety medications administeredin conjunction with the BH4 or BH4-related compound to treat andameliorate autism may include but are not limited to clomipramine,buspirone, buspirone, fluvoxamine, paroxetine, fluoxetine, nefazodone,doxepin, imipramine, bupropion, sertraline, and combinations thereof;mood stabilizing medications administered in conjunction with the BH4 orBH4-related compound to treat and ameliorate autism may include but arenot limited to lithium citrate, valproic acid, lithium carbonate,carbamazepine, and combinations thereof; and an Alzheimer's medicationadministered in conjunction with the BH4 or BH4-related compound totreat and ameliorate autism may include but is not limited to memantineHCl.

In addition, gene therapy with phenylalanine hydroxylase (PAH)(Christensen et al., Mol. Genetics Metabol., 76: 313-318 (2002);Christensen et al., Gene Therapy, 7: 1971-1978 (2000)) or phenylalanineammonia lyase (PAL) (Liu et al., Arts. Cells. Blood. Subs. and Immob.Biotech., 30: 243-257 (2002)) can be employed. Such gene therapytechniques can be used in combination with BH4 therapy and/or dietaryprotein restriction. In other combination therapies, phenylase can beprovided as an injectable enzyme to further lower Phe concentrations inthe patient. Because administration of phenylase would not generatetyrosine (unlike administration of PAH), phenylase treatment would stillresult in tyrosine being an essential amino acid for such patients.Therefore, dietary supplementation with tyrosine may be desired forpatients receiving phenylase in combination with BH4 therapy.

Representative Embodiments

The following representative embodiments of the disclosure are givenmerely to illustrate the disclosure and are not intended to limit thescope of the disclosure.

In one embodiment, provided herein is a stable capsule dosage formcomprising a pharmaceutical formulation comprising an initial amount of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in the crystallineform designated polymorph B, and one or more pharmaceutically acceptableexcipients, wherein the capsule has a shell that is essentially free ofpullulan, and at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or at least about 99.5% ofthe initial amount of the tetrahydrobiopterin dihydrochloride remainsafter the capsule dosage form is stored in a container at about 25° C.and about 60% relative humidity, or at about 40° C. and about 75%relative humidity, for a period of about 3 months, about 4 months, about5 months, about 6 months, about 9 months, about 12 months (one year),about 18 months, about 24 months (two years), about 30 months, or about36 months (three years).

In an embodiment, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 97%, or at least about 99% of the initial amount of thetetrahydrobiopterin dihydrochloride in the stable capsule dosage formdissolves within about 10 minutes, about 15 minutes, about 20 minutes,about 25 minutes, or about 30 minutes after the capsule dosage form hasbeen stored in the container at about 25° C. and about 60% relativehumidity, or at about 40° C. and about 75% relative humidity, for aperiod of about 3 months, about 4 months, about 5 months, about 6months, about 9 months, about 12 months, about 18 months, about 24months, about 30 months, or about 36 months, wherein the dissolution isdetermined according to U.S.P. Method II at 50 r.p.m. in 0.1 Nhydrochloric acid maintained at 37° C.

In a particular embodiment, the container is a heat induction-sealed,screw cap high-density polyethylene (HDPE) bottle. In anotherembodiment, the HDPE bottle is sealed in a foil pouch. In anotherparticular embodiment, the container is a foil blister pack. In anembodiment, the container contains a desiccant. In another embodiment,the container contains no desiccant.

In an embodiment, the initial amount of the tetrahydrobiopterindihydrochloride in the capsule dosage form is in a range from about 100mg to about 500 mg. In specific embodiments, the initial amount of thetetrahydrobiopterin dihydrochloride is about 100 mg, about 150 mg, about160 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, or about 500 mg per capsule.

In one embodiment, the shell of the capsule is made of or comprises oneor more substances selected from the group consisting of cellulosederivatives; hydroxypropyl methylcellulose (HPMC); starch derivatives;carrageenans; acacia; gelatin; polyethylene glycol; homopolymers andcopolymers formed from polyvinyl alcohol, acrylic acid, and methylmethacrylate; and combinations thereof. In a specific embodiment, theshell of the capsule is made of or comprises gelatin. In anotherspecific embodiment, the shell of the capsule is made of or compriseshydroxypropyl methylcellulose.

In one embodiment, provided herein is a stable powder dosage comprisinga pharmaceutical formulation comprising an initial amount of a BH4 orBH4-related compound in pre-blended, powder (i.e., crystalline) form,wherein the powder is pre-mixed and blended to be dissolved in solution.In a specific embodiment, the BH4 or BH4-related compound is(6R)-L-erythro-tetrahydrobiopterin dihydrochloride, designated polymorphB. In a specific embodiment, the pharmaceutical formulation comprises anadditional one or more pharmaceutically acceptable excipients. In aspecific embodiment, the pharmaceutical formulation comprises(6R)-L-erythro-tetrahydrobiopterin dihydrochloride, a sweetener, aflavoring agent, and a flavor enhancer.

In another specific embodiment, the pre-blended powder formulationcomprises the BH4 or BH4-related compound, a sweetener, a flavoringagent, one or more fillers, and a flavor enhancer is a dry blend. In aspecific embodiment, the BH4 or BH4-related compound is(6R)-L-erythro-tetrahydrobiopterin dihydrochloride. In certainembodiments, the formation is prepared by blending the fillers with theBH4 or BH4 compound and flavor enhancer in a blender to achieve aadequate mixture, further blending a portion of the blended mixture withacesulfame potassium or sucralose, a flavoring agent, and ascorbic acidand thereafter passing that mixture through a suitable sieve (e.g., 20mesh sieve), and lastly blending the second mixture with the remainderof the first mixture until the blend is homogenous.

In a specific embodiment, the sweetener is acesulfam potassium, isomalt,Magna Sweet, maltitol, mannitol, sorbitol, sucralose, xylitol, alitmae,neohesperidin dihydrochalcone, trehalose, tagatose, neotame, saccharinand salts thereof, stevioside, erythritol, isomaltulose, polydextrose,luo han guo, monatin, cyclamate, osladine, sucrose, fructose, or glucoseor combinations thereof. In a specific embodiment, the flavor enhanceris anhydrous citric acid, citric acid monohydrate, malic acid, tarticacid, sodium citrate, potassium citrate dehydrate, or sodium potassiumtartate or combinations thereof. In a specific embodiment, the flavoringagent is a cherry, grape, orange, pink lemonade, raspberry, grape,lemon, orange, strawberry, tutti-frutti, tangerine, apple, watermelon,pineapple, banana, peach, kiwi, mango, mixed berry, raspberry lemonade,wild blackberry, blue raspberry, citrus, blueberry, lime, lemon lime,grapefruit, pomegranate, pear, or plum flavors or combinations thereof;and the sieve is a 20 mesh sieve.

In another specific embodiment, the pre-blended powder formulationcomprises 6R)-L-erythro-tetrahydrobiopterin dihydrochloride (i.e., BH4dihydrochloride), the flavor enhancer is citrate, the sweeteners ismannitol, the flavoring agent is orange, and an additional taste maskingagent is sucralose.

In another embodiment, the powder dosage is packaged in a single chambersachet or a dual chamber sachet. In a specific embodiment, the dualchamber sachet separates incompatible components. In a specificembodiment, the dual chamber sachet comprises one chamber that containsthe active BH4 powder blend and a second chamber that contains theflavor blend. In a specific embodiment, the sachets comprise of anadditional active agent for combination therapy.

In another embodiment, the sachets are sealed in Mylar foil pouches. Ina specific embodiment, a desiccant is included in the sachets or in thefoil pouches.

In another embodiment, the amount of the BH4 or BH4 compound in thesachet dosage form is in a range from about 100 mg to about 1300 mg(i.e., 1.3 g). In specific embodiments, the initial amount of thetetrahydrobiopterin dihydrochloride is about 100 mg, about 150 mg, about160 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about1150 mg, about 1200 mg, about 1250 mg, or about 1300 mg per sachet.

In another embodiment, the sachets are filled with 600 mg or more, forexample, of the BH4 or BH4-related compound powder blend wherein theunexpected flowability of the dry blend powder formulation allows forautomated sachet filling.

In another embodiment, the final dosage strength after the active BH4 orBH4 compound from the sachet dosage is dissolved in an aqueous solutionwith the flavor blend is about 10 mg/mL. In specific embodiments, thefinal dosage strength is about 1 mg/mL, about 2 mg/mL, 3 mg/mL, about 4mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL,about 6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about8.5 mg/mL, about 9 mg/mL, about 9.5 mg/mL, about 10 mg/mL, about 10.5mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL,about 15 mg/mL, or about 20 mg/mL.

In a further embodiment, the pharmaceutical formulations in the stablecapsule and sachet dosage forms comprise one or more pharmaceuticallyacceptable excipients selected from the group consisting of ascorbicacid, silicon dioxide, mannitol, microcrystalline cellulose,crospovidone, povidone, stearyl fumaric acid, salt forms of stearylfumarate, dicalcium phosphate, 5-methyltetrahydrofolate (5-MTHF), saltforms of 5-MTHF, and combinations thereof. In an embodiment, the one ormore excipients include crospovidone and stearyl fumaric acid or a saltform of stearyl fumarate. In another embodiment, the one or moreexcipients further include ascorbic acid, silicon dioxide and mannitol.

In a particular embodiment, the pharmaceutical formulation in the stablecapsule comprises an initial amount of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a range from about30% to about 60%, crospovidone from about 3% to about 6%, sodium stearylfumarate from about 1% to about 3%, ascorbic acid from about 1% to about10%, silicon dioxide from about 0.2% to about 2%, and mannitol fromabout 20% to about 50% by weight of the formulation. In anotherparticular embodiment, the pharmaceutical formulation comprises aninitial amount of (6R)-L-erythro-BH4 dihydrochloride from about 40% toabout 50%, ascorbic acid from about 40% to about 50%, crospovidone fromabout 3% to about 6%, sodium stearyl fumarate from about 1% to about 3%,silicon dioxide from about 0.2% to about 2%, and calcium salt of5-methyltetrahydrofolate from about 0.01% to about 0.5% by weight of theformulation.

In another embodiment, the capsule and sachet formulations comprising(6R)-L-erythro-tetrahydrobiopterin dihydrochloride and one or morepharmaceutically acceptable excipients further comprise5-hydroxytryptophan. In a particular embodiment, the formulationcomprises an initial amount of 5-hydroxytryptophan in a range from about20% to about 40% by weight of the formulation.

In yet another embodiment, the stable capsule and sachet dosage forms,including the pharmaceutical formulation therein, are made by a processthat does not include adding liquid water. In an embodiment, thepharmaceutical formulation is made by mixing(6R)-L-erythro-tetrahydrobiopterin dihydrochloride and the one or morepharmaceutically acceptable excipients, without addition of liquidwater. In an embodiment, the pharmaceutical formulation made withoutaddition of liquid water is ground to a powder and packaged in dualchamber sachets. In certain embodiments each sachet is a single dose ormultiple doses. In an embodiment, the pharmaceutical formulation madewithout addition of liquid water is the active ingredient of the stableBH4 capsules.

In a further embodiment, provided herein is a method of treating,ameliorating or preventing hyperphenylalanemia due to BH4 deficiency,comprising administering to a subject in need thereof a therapeuticallyeffective amount of the BH4-containing stable capsule dosage and sachetforms described herein. In one embodiment, the capsule and sachet dosageforms are administered orally once daily. In another embodiment, thecapsule and sachet dosage forms are administered orally twice daily.

In an embodiment, the stable capsule and sachet dosage forms are used totreat, ameliorate or prevent hyperphenylalanemia due to BH4 deficiencywhich is associated with deficiency in or reduced activity of any one orany combination of the enzymes GTP cyclohydrolase 1 (GTP-CH 1),6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR),dihydropteridine reductase (DHPR), and pterin-4-carbinolaminedehydratase (PCD).

In a particular embodiment, the stable capsule and sachet dosage formsused to treat, ameliorate or prevent hyperphenylalanemia due to BH4deficiency comprises a formulation comprising an initial amount of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a range from about30% to about 60%, crospovidone from about 3% to about 6%, sodium stearylfumarate from about 1% to about 3%, ascorbic acid from about 1% to about10%, silicon dioxide from about 0.2% to about 2%, and mannitol fromabout 20% to about 50% by weight of the formulation. In anotherembodiment, the formulation further comprises an initial amount of5-hydroxytryptophan in a range from about 20% to about 40% by weight ofthe formulation.

In an embodiment, the stable capsule and sachet dosage forms are used totreat or ameliorate autism. In a particular embodiment, the stablecapsule and sachet dosage is used to treat or ameliorate autism inchildren. In a particular embodiment, the stable capsule and sachetdosage is used to treat or ameliorate autism in adults. In a particularembodiment, the stable capsule and sachet dosage is used in acombination therapy with an additional medication(s) to treat orameliorate autism.

EXAMPLES

The following examples are provided merely for illustration and are notintended to limit the scope of the disclosure.

Example 1 Dry Process for Making Capsule Dosage Forms

The active ingredient, (6R)-L-erythro-tetrahydrobiopterindihydrochloride or a BH4-related compound, and all the excipients wereseparately pre-screened through an appropriate size mesh (e.g., #20 meshscreen). A powder blend for filling capsules was prepared by mixing theactive ingredient with a glidant (e.g., colloidal silicon dioxide) usinga high shear mixer until a homogenous mix was obtained. The mix wasadded to a suitable blender (e.g., V-blender) and blended with theremaining excipients (e.g., mannitol, ascorbic acid and crospovidone)except for the lubricant. A portion of the powder blend was removed fromthe blender and added to the lubricant (e.g., sodium stearyl fumarate)to obtain a pre-mix. The pre-mix was mixed in a suitable vessel and thenpassed through a screen with appropriate mesh opening (e.g., #20 meshscreen). The resulting material was returned to the blender for a finalmixing. The desired amount of the final powder blend was filled intocapsules (e.g., size 0 or size 00 gelatin or hydroxypropylmethylcellulose capsules) and the capsules were then closed with theircap.

Example 2 Stability of Various Capsule Dosage Forms

Certain embodiments of the capsule dosage forms of the presentdisclosure are illustrated in Table 1, with a comparison tablet dosageform. The (6R)-L-erythro-tetrahydrobiopterin dihydrochloride (BH4-2HCl)in all six capsule examples and the comparison tablet in Table 1 was inthe form of polymorph B. Table 2 summarizes stability data for the Table1 dosage forms in gelatin and HPMC capsules stored in heatinduction-sealed, screw cap high density polyethylene (HDPE) bottles at40° C. and about 75% relative humidity (accelerated stabilityconditions), and comparison data for tablets of Table 1 stored in heatinduction-sealed, HDPE bottles at 40° C. and about 75% relative humidityor stored in foil blister cards under the same conditions. None of thegelatin capsule and HPMC capsule packagings in Table 2 contained adesiccant and the tablet foil blister cards also did not contain adesiccant, while the tablet HDPE bottles contained a desiccant. Table 2demonstrates the ability of various capsule dosage forms to providestable BH4 dihydrochloride over at least six months under theaccelerated stability conditions.

FIGS. 2 and 3 show the dissolution profiles (the rate of dissolution ofBH4 dihydrochloride from the dosage form according to U.S.P. Method IIat 50 r.p.m. in 0.1 N hydrochloric acid at 37° C.) after the capsuledosage forms have been stored at 40° C. and about 75% relative humidity.FIG. 2 shows the dissolution profile of BH4 dihydrochloride in the HPMCcapsule dosage form of Example 3 in Table 1. FIG. 3 shows thedissolution profile of BH4 dihydrochloride in the gelatin capsule dosageform of Example 1 in Table 1. As evident from FIGS. 2 and 3, thedissolution rates of the BH4 dihydrochloride contained in the HPMC andgelatin capsules were maintained over the duration of the stabilitystudies (six months), which is a factor in maintaining thepharmacokinetic properties (e.g., bioavailability) of BH4 over time.

FIG. 4 compares the stability of BH4 capsule dosage forms that have beenstored in heat induction-sealed HDPE bottles or heat induction-sealedHDPE bottles sealed in foil pouches at 40° C. and about 75% relativehumidity, to the stability of BH4 tablets stored in heatinduction-sealed HDPE bottles or foil blister cards under the sameconditions (the stability data are given in Table 2). Storage in HDPEbottles sealed in foil pouches mimics storage in foil blister cards.None of the BH4 capsule packagings contained a desiccant and thetablet-containing foil blister cards also did not contain a desiccant,while the tablet-containing HDPE bottles contained a desiccant. The BH4dihydrochloride in all of these stability studies of the capsules andtablets was in the form of polymorph B.

As demonstrated in Table 2 and FIG. 4, all of the BH4-containing HPMCand gelatin capsule dosage forms exhibited a very small percentage (0.4%or less) of total impurities after being stored without a desiccant for6 months at 40° C. and about 75% relative humidity. As explained above,a capsule dosage form normally is not recommended for a hygroscopic,moisture-sensitive active ingredient such as BH4 dihydrochloride becauseBH4 dihydrochloride would be expected to absorb water from the material(e.g., gelatin or HPMC) in the capsule shell and consequently becomelabile to oxidation. For example, U.S. Pat. No. 4,917,885 teaches thatwhen a hard gelatin capsule is stored under “a highly humid condition,”the active ingredient therein can be “denatured due to the increasedmoisture inside the capsule,” and thus “the quality of a gelatin-madehard capsule is unavoidably degraded in the lapse of time.” Contrary toconventional wisdom, the capsule dosage forms provided herein compriseBH4 dihydrochloride that possesses unexpected stability over a prolongedperiod of time at elevated temperature and high humidity.

TABLE 1 BH4-Containing Formulations of Capsules and Tablets StableCapsule Lots Ingredients wt % Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 TabletBH4•2HCl 46.75 47.13 46.75 46.75 46.75 47.13 33.33 Ascorbic Acidgranular 46.75 47.13 23.38 23.38 0.00 0.00 1.67 Silicon Dioxide 0.750.00 0.75 0.00 0.75 0.00 0.00 Mannitol 0.00 0.00 23.38 24.13 46.75 47.1357.56 Dicalcium phosphate 0.00 0.00 0.00 0.00 0.00 0.00 2.18Crospovidone 4.00 4.00 4.00 4.00 4.00 4.00 4.50 Sodium Stearyl Fumarate1.75 1.75 1.75 1.75 1.75 1.75 0.75 Riboflavin 0.00 0.00 0.00 0.00 0.000.00 0.01 Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0

TABLE 2 Stability of BH4 Capsules and Tablets Stored at 40° C. and about75% Humidity Total Impurity (%) Lot No. Capsule Type Packaging Initial 3months 6 months Ex. 1a Gelatin capsule HDPE bottle 0.23 0.3 0.41 Ex. 2aGelatin capsule 0.22 0.20 0.40 Ex. 3a HPMC capsule 0.18 0.22 0.29 Ex. 3bGelatin capsule 0.19 0.20 0.39 Ex. 4a HPMC capsule 0.20 0.17 0.29 Ex. 4bGelatin capsule 0.21 0.18 0.37 Ex. 5a HPMC capsule 0.26 0.20 0.32 Ex. 5bGelatin capsule 0.24 0.25 0.42 Ex. 6a HPMC capsule 0.26 0.19 0.30 Ex. 6bGelatin capsule 0.26 0.21 0.39 Ex. 1b Gelatin capsule HDPE bottle 0.230.30 0.26 Ex. 2b Gelatin capsule sealed in a 0.22 0.20 0.23 Ex. 3c HPMCcapsule foil pouch 0.18 0.24 0.25 Ex. 3d Gelatin capsule 0.19 0.18 0.25Ex. 4c HPMC capsule 0.20 0.17 0.23 Ex. 4d Gelatin capsule 0.21 0.18 0.23Ex. 5c HPMC capsule 0.26 0.21 0.24 Ex. 5d Gelatin capsule 0.24 0.22 0.24Ex. 6c HPMC capsule 0.26 0.21 0.26 Ex. 6d Gelatin capsule 0.26 0.21 0.23Tablet HDPE bottle 0.25 0.39 0.47 Tablet Foil blister card 0.05 0.290.44

Example 3 HPMC Capsules Containing 160 mg BH4 Dihydrochloride

Table 3 describes the formulation of a stable capsule dosage formcomprising 160 mg (6R)-L-erythro-tetrahydrobiopterin dihydrochloride inpolymorphic form B, wherein the shell of the capsule compriseshydroxypropyl methylcellulose (HPMC).

TABLE 3 Formulation of HPMC Capsules Containing 160 mg BH4•2HClIngredients Function Weight % mg/capsule BH4 Dihydrochloride Active50.00 160.0 Mannitol Diluent 38.50 123.2 Ascorbic Acid GranularAntioxidant 5.00 16.0 Colloidal Silicon Dioxide Glidant 0.75 2.4Crospovidone Disintegrant 4.00 12.8 Sodium Stearyl Fumarate Lubricant1.75 5.6 Total 100.00 320.0

The 160 mg BH4 capsule dosage form of Table 3 was prepared in thefollowing manner. The active ingredient,(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in polymorphic formB, and all the excipients were individually pre-screened through a #20mesh screen. Pre-screened BH4 dihydrochloride and colloidal silicondioxide were dry mixed in a plastic bag for 2 min by shaking. Themixture was screened through a #20 mesh screen, and then mixed at 600rpm for 10 min using a high shear mixer. The resulting mixture wasplaced into a V-blender, and pre-screened ascorbic acid, crospovidoneand mannitol were added. The mixture was mixed for 10 min. Pre-screenedsodium stearyl fumarate was added, and the mixture was mixed for 5 min.The powder blend was passed through a #20 mesh screen. The resultingformulation, having the amounts of ingredients per capsule as shown inTable 3, was filled into size 0 HPMC capsules using a prefill fillingsystem.

HPMC capsules comprising the formulation of Table 3 were stored in heatinduction-sealed, high-density polyethylene (HDPE) bottles containing nodesiccant at 25° C. and about 60% relative humidity (RH) (normalstability conditions), and alternatively at 40° C. and about 75%relative humidity (accelerated stability conditions). Tables 4 and 5show that the 160 mg BH4 HPMC capsules, after storage in HDPE bottlesunder normal and accelerated stability conditions for 3 months,exhibited no significant changes in capsule quality, potency and purity.For example, the capsules contained only 0.26% total impurities after 6months of storage under accelerated stability conditions, i.e., only a0.15% increase in level of total impurities. In addition, FIG. 5demonstrates that the dissolution rate of the 160 mg BH4 dihydrochloridecontained in the HPMC capsules, according to U.S.P. Method II,essentially did not change after the capsules were stored for 1, 3, and6 months under accelerated stability conditions.

TABLE 4 Stability of 160 mg BH4 HPMC Capsules Stored at 25° C. and about60% RH HPMC Capsules Containing 160 mg BH4•2HCl Heat induction-sealedHDPE bottles without desiccant Storage Conditions: 25° C./60% relativehumidity Time Points (months) Test 0 1 3 QUALITY Appearance of capsuleWhite to White to White to off-white off-white off-white capsule capsulecapsule Appearance of capsule Light yellow Light yellow Light yellowcontent powder powder powder POTENCY Assay by HPLC 95.2% 96.5% 101.7% PURITY Related Substances (HPLC) Biopterin ND ND 0.02% DihydrobiopterinND ND 0.03% R-Tetrahydrobiolumazine 0.03% 0.04% 0.05%S-Tetrahydrobiopterin 0.03% 0.02% 0.04% Tetrahydropterin 0.05% 0.04%0.06% Individual Unidentified ND ND ND Total Unidentified ND ND ND Total0.11% 0.10% 0.20% ND = Not Detected RRT = Relative Retention Time

TABLE 5 Stability of 160 mg BH4 HPMC Capsules Stored at 40° C. and about75% RH HPMC Capsules Containing 160 mg BH4•2HCl Heat induction-sealedHDPE bottles without desiccant Storage Conditions: 40° C./75% relativehumidity Time Points (months) Test 0 1 3 QUALITY Appearance of capsuleWhite to White to White to off-white off-white off-white capsule capsulecapsule Appearance of capsule Light yellow Light yellow Light yellowcontent powder powder powder POTENCY Assay by HPLC 95.2% 99.0% 98.7%PURITY Related Substances (HPLC) Biopterin ND 0.02% 0.05%Dihydrobiopterin ND 0.02% 0.06% R-Tetrahydrobiolumazine 0.03% 0.04%0.06% S-Tetrahydrobiopterin 0.03% 0.02% 0.03% Tetrahydropterin 0.05% ND0.06% Individual Unidentified ND ND ND Total Unidentified ND ND ND Total0.11% 0.10% 0.26% ND = Not Detected RRT = Relative Retention Time

Example 4 HPMC Capsules Containing 200 mg BH4 Dihydrochloride

Table 6 describes the formulation of a stable capsule dosage formcomprising 200 mg (6R)-L-erythro-tetrahydrobiopterin dihydrochloride inpolymorphic form B, wherein the shell of the capsule compriseshydroxypropyl methylcellulose (HPMC). This capsule dosage form wasprepared in a manner similar to the preparative procedure described inExample 3.

TABLE 6 Formulation of HPMC Capsules Containing 200 mg BH4•2HClIngredients Function Weight % mg/capsule BH4 Dihydrochloride Active50.00 200.0 Mannitol Diluent 41.00 164.0 Ascorbic Acid Fine PowderAntioxidant 2.50 10.0 Colloidal Silicon Dioxide Glidant 0.75 3.0Crospovidone Disintegrant 4.00 16.0 Sodium Stearyl Fumarate Lubricant1.75 7.0 Total 100.00 400.0

HPMC capsules comprising the formulation of Table 6 were stored in heatinduction-sealed, HDPE bottles containing no desiccant at 40° C. andabout 75% relative humidity. As evident from Table 7, the 200 mg BH4HPMC capsules showed no significant changes in capsule quality, potencyand purity after being stored in HDPE bottles under acceleratedstability conditions for 6 months. For example, the capsules containedonly 0.29% total impurities after 6 months of storage under acceleratedstability conditions, which was only a 0.1% increase in level of totalimpurities. Further, FIG. 6 shows that the dissolution profile of the200 mg BH4 dihydrochloride contained in the HPMC capsules, according toU.S.P. Method II, did not significantly change after the capsules werestored for 3 months under accelerated stability conditions.

TABLE 7 Stability of 200 mg BH4 HPMC Capsules Stored at 40° C. and about75% RH HPMC Capsules Containing 200 mg BH4•2HCl Heat induction-sealedHDPE bottles without desiccant Storage Conditions: 40° C./75% relativehumidity Time Points (months) Test 0 3 6 QUALITY Appearance of capsuleWhite to White to White to off-white off-white off-white capsule capsulecapsule Appearance of capsule Light yellow Light yellow Light yellowcontent powder powder POTENCY Assay by HPLC 92.7% 93.1% 91.2% PURITYRelated Substances (HPLC) Biopterin ND 0.03% 0.06% Dihydrobiopterin0.03% 0.05% 0.06% R-Tetrahydrobiolumazine 0.05% 0.05% 0.06%S-Tetrahydrobiopterin 0.04% 0.03% 0.03% Tetrahydropterin 0.06% 0.03%0.07% Individual Unidentified 0.04% 0.03% 0.005%  (RRT = 1.3) (RRT =1.3) Total Unidentified ND ND 0.005%  Total 0.18% 0.19% 0.29% ND = NotDetected RRT = Relative Retention Time

The effect of storage with a desiccant on the stability of BH4dihydrochloride was also evaluated. HPMC capsules comprising theformulation of Table 6 were stored in heat induction-sealed HDPE bottlescontaining varying amounts of silica gel desiccant for 6 months at 40°C. and about 75% relative humidity. As evident from Table 8, storage ofthe HPMC capsules in HDPE bottles containing increasing amounts ofsilica gel desiccant under accelerated stability conditions had noeffect on the stability of BH4 dihydrochloride. With or without adesiccant, the 200 mg BH4 HPMC capsules contained a slightly higheramount of total impurities (0.29%) after 6 months of storage underaccelerated stability conditions as they did prior to storage (0.18%).

TABLE 8 Effect of Desiccant on Stability of BH4 HPMC Capsules Stored at40° C./75% RH HPMC Capsules Containing 200 mg BH4•2HCl Heatinduction-sealed HDPE bottles with varying amounts of silica geldesiccant Storage Conditions: 40° C./75% relative humidity Time (month)and Amount (g) of Desiccant 3 mo 3 mo 3 mo 6 mo 6 mo 6 mo Test 0 mo 0 g1 g 2 g 0 g 1 g 2 g Quality Appearance of capsule White to White toWhite to White to White to White to White to off-white off-whiteoff-white off-white off-white off-white off-white Appearance of capsuleLight Light Light Light Light Light Yellow content yellow yellow yellowyellow yellow yellow Purity Related Substances (HPLC) Biopterin ND 0.03%0.02% 0.03% 0.06% 0.04% 0.05% Dihydrobiopterin 0.03% 0.05% 0.06% 0.07%0.06% 0.07% 0.07% R-Tetrahydrobiolumazine 0.05% 0.05% 0.06% 0.04% 0.06%0.05% 0.05% S-Tetrahydrobiopterin 0.04% 0.03% 0.03% 0.02% 0.03% 0.03%0.04% Tetrahydropterin 0.06% 0.03% 0.02% 0.02% 0.07% 0.08% 0.07%Individual Unidentified ND ND ND ND 0.005%  0.005%  0.009%  (RRT = 0.5)(RRT = 0.5) 0.005%  0.006%  (RRT = 0.6) (RRT = 0.6) Total UnidentifiedND ND ND ND 0.005%  0.01% 0.015%  Total 0.18% 0.19% 0.19% 0.18% 0.29%0.28% 0.29% ND = Not Detected RRT = Relative Retention Time

The dissolution profile of the 200 mg BH4 dihydrochloride contained inthe HPMC capsules, according to U.S.P. Method II, slightly differeddepending on whether the capsules were stored in HDPE bottles with orwithout a desiccant for 3 months under accelerated stability conditions.FIG. 7 shows that storage with silica gel desiccant slightly decreasedthe dissolution lag time of the BH4 dihydrochloride and slightly sped upits dissolution.

TABLE 9 HPMC Capsules Containing 200 mg BH4 Dihydrochloride withSucralose Ingredients Function Weight % mg/capsule BH4 DihydrochlorideActive 50.00 200.0 Mannitol Diluent 37.00 148.0 Ascorbic Acid FinePowder Antioxidant 2.50 10.0 Colloidal Silicon Dioxide Glidant 0.75 3.0Sucralose Sweetener 4.00 16.0 Crospovidone Disintegrant 4.00 16.0 SodiumStearyl Fumarate Lubricant 1.75 7.0 Total 100.00 400.0

HPMC capsules comprising the formulation of Table 9 were stored in heatinduction-sealed, HDPE bottles with varying amounts of silica geldesiccant at 40° C. and about 75% relative humidity. As evident fromFIG. 9, storage of the HPMC capsules in HDPE bottles containingincreasing amounts of silica gel desiccant under accelerated stabilityconditions is detrimental to the physical stability of BH4dihydrochloride; black specks were observed in the powder content of thecapsules stored with 1 g and 2 g of desiccant after 6 months. Stabilityof the capsule stored without desiccant remains acceptable after 6months of storage at 40° C./&5% RH. As evident from Table 10, the 200 mgBH4 HPMC capsules without desiccant showed insignificant change incapsule quality, potency and purity after being stored in HDPE bottlesunder accelerated stability conditions for 6 months. For example, thecapsules contained only 0.29% total impurities after 6 months of storageunder accelerated stability conditions, which was only about 0.1%increase in level of total impurities.

TABLE 10 Stability of BH4 HPMC Capsules (with sucralose) Stored at 40°C./75% RH HPMC Capsules Containing 200 mg BH4•2HCl Heat induction-sealedHDPE bottles without silica gel desiccant Storage Conditions: 40° C./75%relative humidity Time (month) Test 0 mo 3 mo 6 mo Quality Appearance ofcapsule White to White to White to off-white off-white off-whiteAppearance of capsule Light yellow Light yellow Yellow content PotencyBH4 95.1% 92.2% 94.1% Purity Related Substances (HPLC) Biopterin ND0.03% 0.06% Dihydrobiopterin 0.03% 0.06% 0.06% R-Tetrahydrobiolumazine0.05% 0.05% 0.06% S-Tetrahydrobiopterin 0.04% 0.02% 0.03%Tetrahydropterin 0.06% 0.03% 0.07% Individual Unidentified ND ND 0.003% (RRT = 0.51) 0.005%  (RRT = 0.63) Total Unidentified ND ND 0.008%  Total0.18% 0.19% 0.29%

Example 5 HPMC Capsules Containing 250 mg BH4 Dihydrochloride

Table 11 describes the formulation of a stable capsule dosage formcomprising 250 mg (6R)-L-erythro-tetrahydrobiopterin dihydrochloride inpolymorphic form B, wherein the shell of the capsule compriseshydroxypropyl methylcellulose (HPMC). This capsule dosage form wasprepared in a manner similar to the preparative procedure described inExample 3, wherein the order of addition of ingredients was BH4dihydrochloride and silicon dioxide, followed by ascorbic acid andcrospovidone, then followed by 5-methyltetrahydrofolate, and finallyfollowed by sodium stearyl fumarate.

TABLE 11 Formulation of HPMC Capsules Containing 250 mg BH4•2HClIngredients Function Weight % mg/capsule BH4 Dihydrochloride Active46.75 250.0 Ascorbic Acid Granular Antioxidant 46.75 250.0 ColloidalSilicon Dioxide Glidant 0.75 4.0 Crospovidone Disintegrant 3.96 21.2Sodium Stearyl Fumarate Lubricant 1.75 9.4 5-Methyltetrahydrofolate,Dietary 0.04 0.2 Calcium Salt Supplement Total 100.00 534.8

HPMC capsules comprising the formulation of Table 11 were stored in heatinduction-sealed, high-density polyethylene (HDPE) bottles containing nodesiccant at 25° C. and about 60% relative humidity (normal stabilityconditions), and alternatively at 40° C. and about 75% relative humidity(accelerated stability conditions). Tables 12 and 13 show that the 250mg BH4 HPMC capsules, after storage in HDPE bottles under normal andaccelerated stability conditions for 3 month, displayed no significantchanges in capsule quality, potency and purity. For example, thecapsules contained only 0.25% total impurities after 3 month of storageunder normal stability conditions, which was only a 0.15% increase inlevel of total impurities, and the capsules contained only 0.0.3% totalimpurities after 3 month of storage under accelerated stabilityconditions, which was only an increase of 0.2% total impurities.

TABLE 12 Stability of 250 mg BH4 HPMC Capsules Stored at 25° C. andabout 60% RH HPMC Capsules Containing 250 mg BH4•2HCl Heatinduction-sealed HDPE bottles without desiccant Storage Conditions: 25°C./60% relative humidity Time Points (months) Test 0 1 3 QUALITYAppearance of capsule White to White to White to off-white off-white offwhite capsule capsule capsule Appearance of capsule Light yellow Lightyellow Light yellow content powder powder powder POTENCY Assay by HPLC99.7% 101.3%  102.3%  PURITY Related Substances (HPLC) Biopterin ND0.01% 0.02% Dihydrobiopterin ND 0.02% 0.04% R-Tetrahydrobiolumazine0.04% 0.03% 0.05% S-Tetrahydrobiopterin 0.03% 0.02% 0.04%Tetrahydropterin 0.03% ND 0.10% Individual Unidentified ND 0.03% ND (RRT= 0.72) Total Unidentified ND 0.03% ND Total 0.10% 0.11% 0.25% ND = NotDetected RRT = Relative Retention Time

TABLE 13 Stability of 250 mg BH4 HPMC Capsules Stored at 40° C. andabout 75% RH HPMC Capsules Containing 250 mg BH4•2HCl Heatinduction-sealed HDPE bottles without desiccant Storage Conditions: 40°C./75% relative humidity Time Points (months) Test 0 1 3 QUALITYAppearance of capsule White to White to White to off-white off-whiteoff-white capsule capsule capsule Appearance of capsule Light yellowLight yellow Light yellow content powder powder powder POTENCY Assay byHPLC 99.7% 96.8% 101.9%  PURITY Related Substances (HPLC) Biopterin ND0.02% 0.04% Dihydrobiopterin ND 0.03% 0.07% R-Tetrahydrobiolumazine0.04% 0.03% 0.06% S-Tetrahydrobiopterin 0.03% 0.02% 0.04%Tetrahydropterin 0.03% ND 0.09% Individual Unidentified ND 0.04% ND (RRT= 0.72) Total Unidentified ND 0.04% ND Total 0.10% 0.14% 0.30% ND = NotDetected RRT = Relative Retention Time

Example 6 Clinical Evaluation of BH4 Capsules

In designing a clinical evaluation of (6R)-L-erythro-tetrahydrobiopterindihydrochloride, an important consideration is that BH4 is an essentialco-factor for two enzymes primarily located in the central nervoussystem (CNS), tyrosine and tryptophan hydroxylases, and the dose of BH4needed for normal neurotransmitter metabolism in the CNS issignificantly higher than the dose needed to improve PAH activity in theliver.

The clinical evaluation comprises two clinical studies. In both studies,BH4 dihydrochloride is administered once daily to achieve higher peakconcentrations and consequently improved CNS penetration.

A first study evaluates the relative bioavailability of BH4dihydrochloride administered as a capsule in fasted conditions and aftera meal in healthy subjects. Ascending doses of BH4 dihydrochloride up to40 mg/kg/day are evaluated and drug levels in cerebrospinal fluid (CSF)are measured. Administration of higher doses up to 40 mg/kg/day as asingle dose is expected to achieve higher peak levels of BH4 in CSF,provide availability of BH4 in CSF, and improve penetration of BH4 intothe CNS. Measurement of BH4 levels in CSF is expected to be indicativeof BH4 availability in brain tissues.

A second study evaluates the safety and efficacy of BH4 dihydrochloridein subjects with hyperphenylalaninemia due to BH4 deficiency. Thesubjects continue to receive the maximum dose of BH4 dihydrochloridetolerated in the first study for a total of 3 months. The primaryendpoint for the second study is a measure of blood phenylalaninecontrol. Moreover, other CSF parameters and neurological outcomes areevaluated. For example, serotonin and folate metabolism can be monitoredby assessing 5-HIAA and 5-MTHF levels in the CSF. Further, becausedopamine inhibits the secretion of prolactin and hyperprolactinemia hasbeen documented in patients with BH4 deficiency, change in prolactinconcentrations can function as a useful indicator of dopamine synthesisand content in the brain.

Example 7 Dry Process for Making Sachet Dosage Forms

The active ingredient, (6R)-L-erythro-tetrahydrobiopterindihydrochloride or a BH4-related compound, and all the excipients wereseparately pre-screened through an appropriate size mesh (e.g., #20 meshscreen). A powder blend for sachets was prepared by adding one-half ofthe preweighed sweetener to a suitable blender (e.g., V-blender), addingthe preweighed BH4 or a BH4-related compound to the blender, adding thepreweighed flavor enhancer to the blender, and adding the remainingsweetener to the blender. The dry mixture was blended until the it wasadequately mixed. A portion of the first mixture was then mixed with apreweighed amount of acesulfame potassium or sucralose, a flavoringagent, and ascorbic acid in a separate blender. The second mixture waspassed through a suitable sieve (e.g., #20 mesh sieve) and then added tothe remainder of the first mixture in the first blender and mixed untilthe blend was homogonous. The desired amount of the final powder blendwas filled into sachets (e.g., single or double chamber sachets).

The sachets can be flushed with inert gas. The sachets can behermetically sealed. The sachets can include a desiccant. The sachetsmay be further packaged in a Mylar pouch. The Mylar pouch may contain adesiccant.

Example 8 Sachet Dosage Forms

Certain embodiments of the sachet dosage forms of the present disclosureare illustrated in Tables 14, 16, and 17. The sapropterindihydrochloride in all seven sachet examples in comparison Table 12 wasin the form of polymorph B. Table 12 gives examples of formulations thatcomprise an amount of BH4 dihydrochloride of about 15% and between75-80% mannitol, about 0-1% sucralose, about 1.5% flavoring agent,between 4.5-7% potassium citrate or potassium sodium tartrate, and about1% ascorbic acid fine powder. In a specific embodiment, the flavoringagent is strawberry, orange on a sucrose substrate, orange on a mannitolsubstrate, or grape. In another specific embodiment, the blend alsoconsists of about 5% potassium citrate.

Certain embodiments of the stability of the dosage forms from Table 12are provided in Table 15. Table 15 presents stability data oncompositions F, C1, and C2 from Table 12. Stability under differentstorage conditions were assessed and the potency of the active BH4dihydrochloride is presented.

In an additional embodiment, the pharmaceutical formulation in thestable sachet dosage forms comprise an amount of BH4 dihydrochloride ofabout 15.1% BH4 dihydrochloride, 75.3% mannitol, 1.5% orange flavor on asucrose base, 0.8% sucralose, 4.8% potassium citrate, and 0.8% ascorbicacid fine powder (Table 16).

TABLE 14 Examples of BH4 Dihydrochloride Dry blend Powder FormulationsPercent (weight/weight) Ingredient A B C1 C2 D E F Sapropterindihydrochloride 15.6  15.1  15.3  15.3  15.1  15.1  15.1  Mannitol 77.9 35.0  76.7  76.7  75.2  — 75.2  Sorbitol — — — — — 75.2  — Xylitol —40.8  — — — — — Acesulfame potassium — 0.2 — — Sucralose 0.8 — 0.8 0.80.8 0.8 0.8 Strawberry flavor — 1.5 — — — 1.5 Orange flavor on sucrose —— 1.5 — — — — substrate Orange flavor on mannitol — — — 1.5 — — —substrate Grape flavor — 1.5 1.5 — Potassium Citrate 4.9 — 4.9 4.9 — —Potassium sodium tartrate — 6.6 — — 6.6 6.6 6.6 Ascorbic Acid Finepowder 0.8 0.8 0.8 0.8 0.8 0.8 0.8 — — — — — — — Total (g) 100.0  100.0 100.0  100.0  100.0  100.0  100.0 

In an additional embodiment, the pharmaceutical formulation in thestable sachet dosage forms comprise an amount of BH4 dihydrochloride ofabout 15.3% BH4 dihydrochloride, 76.7% mannitol, 1.5% orange flavor on amannitol base, 0.8% sucralose, 4.8% potassium citrate, and 0.8% ascorbicacid fine powder (Table 16).

In another embodiment, the pharmaceutical formulation in the stablesachet dosage forms comprise a total amount of 0.835 g dry powder blendwith the amount of BH4 dihydrochloride about 23.9% of the weight or 0.2g (Table 17).

TABLE 15 Stability of BH4 Dihydrochloride FormulationsDegradation/impurity (area %) Potency R-Tetrahydro- S-Tetrahydro-Individual Total Total Formulation (%) Biopterin BH2^(a) biolumazinebiolumazine S-BH4^(b) BH4^(b) unidentified unidentified Impurities (%)Storage Condition: Initial time point 100 μg/mL 100.6 ND 0.04 0.05 ND^(c) 0.03 0.05 ND ND 0.17 reference API F 104.7 0.10 0.05 0.05 ND0.04 0.05 ND ND 0.29 C1 101.9 0.01 0.03 0.05 ND 0.04 0.05 ND ND 0.18 C2105.8 0.02 0.04 0.05 ND 0.04 0.06 ND ND 0.21 Storage Condition: 1 monthat 25° C./60% RH C1 94.8 0.02 0.04 0.04 ND 0.03 0.05 ND ND 0.18 C2 98.00.02 0.04 0.05 ND 0.04 0.05 ND ND 0.20 Storage Condition: 1 month at 40°C./75% RH F 97.2 0.20 0.06 0.37 0.01 0.03 0.04 0.01  0.01  0.77 0.05  C191.8 0.029 0.132 0.038 ND ND ND 0.062 0.062 0.26 C2 97.7 0.026 0.1230.082 ND ND ND ND ND 0.23 Storage Condition: 3 months at 40° C./75% RHC1 48.48 0.106 0.162 0.093 ND  0.009  0.057 0.012 0.203 0.63 0.030 0.0180.079 0.064 C2 96.37 0.138 0.304 0.154 0.057 0.01 0.105 0.036 0.135 0.900.068 0.013 0.018 ^(a)BH2 = Dihydrobiopterin; ^(b)BH4 =Tetrahydrobiopterin; and ^(c)ND = Not Detected;

TABLE 16 Examples of BH4 Dihydrochloride Dry blend Powder FormulationsExample 1 Example 2 Ingredient Function % w/w BH4 Active 15.1  15.3 Mannitol Sweetener 75.3  76.7  Orange Flavor (Sucrose base) Flavor 1.5 —Orange Flavor (Mannitol base) Flavor — 1.5 Sucralose Sweetener 0.8 0.8Potassium Citrate Flavor 4.8 4.8 Enhancer Ascorbic Acid Fine Flavor 0.80.8 Enhancer Total 100% 100%

TABLE 17 Examples of Sachet Dosage Formulations of BH4 DihydrochlorideFormulation C Formulation D Formulation E Ingredient g/sachet % g/sachet% g/sachet % Sapropterin 0.2000 23.95 0.2000 23.95 0.2000 23.95 HClMannitol 0.3400 40.72 0.3381 40.49 0.3369 40.34 Sucralose 0.0100 1.200.0119 1.42 0.0131 1.57 Potassium 0.0650 7.78 0.0650 7.78 0.0650 7.78Citrate Ascorbic Acid 0.0100 1.20 0.0100 1.20 0.0100 1.20 Citric acid0.1500 17.96 0.1500 17.96 0.1500 17.96 anhydrous Sodium citrate 0.06007.19 0.0600 7.19 0.0600 7.19 dihydrate Total 0.8350 100.00 0.8350 100.000.8350 100.00

Example 9 Stability of Various Sachet Dosage Forms

Certain embodiments of the sachet dosage forms of the present disclosureare illustrated in Table 18, with a comparison of the stability ofdifferent BH4 or BH4-related compounds packaged in foil pouches.

TABLE 18 Stability of BH4 Dihydrochloride Sachet Packaged in FoilPouches T = 1 Month T = 3 Month Lot Lot Lot Lot 11434-50 11434-5111434-50 11434-51 Orange Orange Orange Orange 40° C./75% RH (Sucrose(Mannitol (Sucrose (Mannitol Sample base) base) base) base) Biopterin(RRT = .5) % 0.029 0.026 0.106 0.138 BH2 (RRT = .6) % 0.132 0.123 0.1620.304 R-THBL (RRT = .79) % 0.038 0.082 0.093 0.154 S-THBL (RRT = .88) %ND ND ND 0.057 BH4 % 91.75 97.74 48.48 96.37 S-BH4 (RRT = 1.2) % ND ND0.009 0.010 THP (RRT = 1.4) % ND ND 0.057 0.105 Unknown Impurity ND ND0.012 0.036 (RRT = 1.4) % Unknown Impurity ND ND 0.030 0.068 (RRT = .54)% Unknown Impurity ND ND 0.018 0.013 (RRT = .58) % Unknown Impurity0.062 ND 0.079 0.018 (RRT = .72) % Unknown Impurity ND ND 0.064 ND (RRT= .81) % Total Unknown Impurity 0.062 ND 0.203 0.135 Total Impurity0.261 0.231 0.629 0.903 ND = Not Detected

Example 10 Sachet Dosage Form Data without Flavoring

Certain embodiments of the sachet dosage forms of the present disclosureare illustrated in Table 19, wherein the sachet formulation may consistof a BH4 or BH4-related compound, a sweetener, and flavor enhancerswithout a flavoring agent.

It is understood that every embodiment described herein can optionallybe combined with any one or more of the other embodiments describedherein.

Every patent literature and every non-patent literature cited herein areincorporated herein by reference in their entirety.

TABLE 19 Sachet dosage formulation without flavoring agents Ingredientg/sachet % Sapropterin HCl 0.2 32 Mannitol 0.35 56 Potassium 0.065 10.4Citrate Ascorbic Acid 0.01 1.6 TOTAL 0.625 100

Numerous modifications and variations to the disclosure, as set forth inthe embodiments and illustrative examples described herein, are expectedto occur to those skilled in the art. Consequently, only suchlimitations as appear in the accompanying claims should be placed on thedisclosure.

Example 11 Sachet Dosage Form Containing 200 mg BH4 Dihydrochloride

Table 20 describes the formulation of a stable sachet dosage formcomprising 200 mg (6R)-L-erythro-tetrahydrobiopterin dihydrochloride inpolymorphic form B. This sachet dosage form was prepared in a manner asprovided in the Detailed Description.

TABLE 20 Sachet dosage formulation containing 200 mg BH4 DihydrochlorideIngredient mg/sachet % Sapropterin 200 32 2HCl Sucralose 11.9 1.9micronized Potassium citrate 65 10.4 monohydrate Ascorbic acid 10 1.6fine powder TOTAL 625 100

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the claimed embodiments, and are not intended to limit thescope of what is disclosed herein. Modifications that are obvious topersons of skill in the art are intended to be within the scope of thefollowing claims. All publications, patents, and patent applicationscited in this specification are incorporated herein by reference as ifeach such publication, patent or patent application were specificallyand individually indicated to be incorporated herein by reference.

What is claimed is:
 1. A pharmaceutical composition, comprising a dryblend powder of a BH4 or BH4-related compound and an excipient.
 2. Thepharmaceutical composition of claim 1, wherein the amount of the BH4 orBH4-related compound in the composition is between about 5% and about55% by weight.
 3. The pharmaceutical composition of claim 1, wherein theexcipient is a sweetening agent selected from the group consisting ofacesulfam potassium, isomalt, Magna Sweet, maltitol, mannitol, sorbitol,sucralose, xylitol, alitmae, neohesperidin dihydrochalcone, trehalose,tagatose, neotame, saccharin and salts thereof, stevioside, erythritol,isomaltulose, polydextrose, luo han guo, monatin, cyclamate, osladine,sucrose, fructose, and glucose, and combinations thereof.
 4. Thepharmaceutical composition of claim 1, wherein the excipient is aflavoring agent selected from the group consisting of cherry, grape,orange, pink lemonade, raspberry, grape, lemon, orange, strawberry,tutti-frutti, tangerine, apple, watermelon, pineapple, banana, peach,kiwi, mango, mixed berry, raspberry lemonade, wild blackberry, blueraspberry, citrus, blueberry, lime, lemon lime, grapefruit, pomegranate,pear, and plum flavors, and combinations thereof.
 5. The pharmaceuticalcomposition of claim 1, wherein the excipient is a flavor enhancerselected from the group consisting of anhydrous citric acid, citric acidmonohydrate, malic acid, tartic acid, sodium citrate, potassium citratedihydrate, sodium potassium tartate, ascorbic acid, and sodiumascorbate, and combinations thereof.
 6. The pharmaceutical compositionof claim 1, wherein the excipient is a filler selected from the groupconsisting of isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol,sucrose, and fructose, and combinations thereof.
 7. The pharmaceuticalcomposition of claim 1, wherein the dry blend powder comprises about 15%to about 30% of the BH4 or BH4-related compound by weight.
 8. Thepharmaceutical composition of claim 1, wherein the dry blend powdercomprises about 30% to about 50% of the BH4 or BH4-related compound byweight.
 9. The pharmaceutical composition of claim 1, wherein the BH4 orBH4-related compound is (6R)-L-erythro-tetrahydrobiopterindihydrochloride.
 10. The pharmaceutical composition of claim 1, whereinthe BH4 or BH4-related compound is the polymorph B form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.
 11. Thepharmaceutical composition of claim 1, wherein the BH4 or BH4-relatedcompound is packaged in a hermetically sealed container.
 12. Thepharmaceutical composition of claim 1, wherein the BH4 or BH4-relatedcompound is packaged in a non-hermetically sealed container.
 13. Asingle chamber sachet dosage form, comprising the composition ofclaim
 1. 14. A dual chamber sachet dosage form, comprising thecomposition of claim 1 in one chamber and a dry flavor blend in theother chamber.
 15. The sachet dosage form of claim 13, furthercomprising a desiccant.
 16. The sachet dosage form of claim 13, whereinthe dosage is mixed with a liquid prior to ingestion.
 17. The sachetdosage form of claim 13, wherein the dosage comprises about 5%, about7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about22.5%, about 25%, about 27.5%, about 30%, about 32.5%, about 35%, about37.5%, about 40%, about 42.5%, about 45%, about 47.5%, about 50%, about52.5%, or about 55% BH4 dihydrochloride by weight.
 18. The sachet dosageform of claim 13, wherein the dosage comprises between about 50 mg andabout 1300 mg of the BH4 or BH4-related compound.
 19. The sachet dosageform of claim 13, wherein the dosage comprises an additionalpharmaceutical formulation.
 20. The sachet dosage form of claim 13,wherein the pharmaceutical formulation produces a clear solution whendissolved in an aqueous solution.
 21. The sachet dosage form of claim13, wherein the sachet is a hermetically sealed sachet.
 22. The sachetdosage form of claim 13, wherein the dosage comprises about 32% BH4dihydrochloride, about 55.1% mannitol, about 1.9% sucralose micronized,about 10.4% potassium citrate monohydrate, and about 1.6% ascorbic acidfine powder by weight.
 23. The sachet dosage form of claim 13, whereinthe dosage comprises about 32% BH4 dihydrochloride, about 55.4%mannitol, about 1.6% sucralose, about 10.4% potassium citrate, and about1.6% ascorbic acid by weight.
 24. The sachet dosage form of claim 13,wherein the dosage comprises about 200 mg BH4 dihydrochloride, about 338mg mannitol, about 12 mg sucralose micronized, about 65 mg potassiumcitrate monohydrate, and about 10 mg ascorbic acid fine powder.
 25. Thesachet dosage form of claim 13, wherein the dosage comprises about 100mg BH4 dihydrochloride.
 26. The sachet dosage form of claim 13, whereinno less than about 90% of the initial amount of the BH4 or BH4-relatedcompound is present after 3 months at 40° C. and 75% Relative Humidity.27. The sachet dosage form of claim 13, wherein no less than about 90%of the initial amount of the BH4 or BH4-related compound is presentafter 2 years at room temperature.
 28. The sachet dosage form of claim13, wherein at least about 90% of the initial amount of the BH4 orBH4-related compound remains, and wherein at least about 85% of theinitial amount of the BH4 or BH4-related compound dissolves within about15 minutes, after the sachet dosage form is stored at about 40° C. andabout 75% RH for a period of about three months.
 29. A stable capsuledosage form comprising a pharmaceutical formulation comprising aninitial amount of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride ina crystalline form designated polymorph B, and one or morepharmaceutically acceptable excipients, wherein: a. the capsule has ashell that is essentially free of pullulan, and b. at least about 98% ofthe initial amount of the tetrahydrobiopterin dihydrochloride remainsafter the capsule dosage form is stored in a container at about 40° C.and about 75% relative humidity for a period from about three months toabout six months.
 30. The stable capsule dosage form of claim 29,wherein the container is a heat induction-sealed, screw cap high-densitypolyethylene bottle.
 31. The stable capsule dosage form of claim 29,wherein the container contains no desiccant.
 32. The stable capsuledosage form of claim 29, wherein the initial amount of thetetrahydrobiopterin dihydrochloride in the capsule dosage form is in arange from about 100 mg to about 500 mg.
 33. The stable capsule dosageform of claim 29, wherein the initial amount of the tetrahydrobiopterindihydrochloride in the capsule dosage form is about 150 mg, or about 160mg, or about 200 mg, or about 250 mg, or about 300 mg per capsule. 34.The stable capsule dosage form of claim 29, wherein the shell of thecapsule comprises one or more substances selected from the groupconsisting of cellulose derivatives; hydroxypropyl methylcellulose;starch derivatives; carrageenans; acacia; gelatin; polyethylene glycol;homopolymers and copolymers formed from polyvinyl alcohol, acrylic acid,and methyl methacrylate; and combinations thereof.
 35. The stablecapsule dosage form of claim 29, wherein the shell of the capsulecomprises gelatin or hydroxypropyl methylcellulose.
 36. The stablecapsule dosage form of claim 29, wherein the one or more excipients areselected from the group consisting of ascorbic acid, silicon dioxide,mannitol, microcrystalline cellulose, crospovidone, povidone, stearylfumaric acid, salt forms of stearyl fumarate, dicalcium phosphate, and5-methyltetrahydrofolate (5-MTHF), and salt forms thereof.
 37. Thestable capsule dosage form of claim 29, wherein the one or moreexcipients comprise crospovidone, and stearyl fumaric acid or a saltform of stearyl fumarate.
 38. The stable capsule dosage form of claim29, wherein the one or more excipients further comprise ascorbic acid,silicon dioxide, and mannitol.
 39. The stable capsule dosage form ofclaim 29, wherein the pharmaceutical formulation comprises an initialamount of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a rangefrom about 30% to about 60%, crospovidone from about 3% to about 6%,sodium stearyl fumarate from about 1% to about 3%, ascorbic acid fromabout 1% to about 10%, silicon dioxide from about 0.2% to about 2%, andmannitol from about 20% to about 50% by weight of the formulation. 40.The stable capsule dosage form of claim 29, wherein the pharmaceuticalformulation further comprises 5-hydroxytryptophan.
 41. The stablecapsule dosage form of claim 29, wherein the pharmaceutical formulationcomprises an initial amount of 5-hydroxytryptophan in a range from about20% to about 40% by weight of the formulation.
 42. The stable capsuledosage form of claim 29, wherein the pharmaceutical formulationcomprises an initial amount of (6R)-L-erythro-BH4 dihydrochloride fromabout 40% to about 50%, ascorbic acid from about 40% to about 50%,crospovidone from about 3% to about 6%, sodium stearyl fumarate fromabout 1% to about 3%, silicon dioxide from about 0.2% to about 2%, andcalcium salt of 5-methyltetrahydrofolate from about 0.01% to about 0.5%by weight of the formulation.
 43. The stable capsule dosage form ofclaim 29, wherein the pharmaceutical formulation is made by mixing thetetrahydrobiopterin dihydrochloride and the one or more pharmaceuticallyacceptable excipients, without addition of liquid water.
 44. The stablecapsule dosage form of claim 29, wherein the dosage is useful forreducing blood phenylalanine levels in patients withhyperphenylalaninemia due to tetrahydrobiopterin-responsephenylketonuria.
 45. The stable capsule dosage form of claim 29, whereinthe dosage is useful for reducing blood phenylalanine levels in patientswith hyperphenylalaninemia due to tetrahydrobiopterin-responsephenylketonuria in conjunction with a phenylalanine restricted diet. 46.The stable capsule dosage form of claim 29, wherein the dosage is usefulfor treating or ameliorating conditions associated with elevatedphenylalanine levels or decreased tyrosine or tryptophan levels.
 47. Thestable capsule dosage form of claim 29, wherein the dosage is useful fortreating or ameliorating autism.
 48. The stable capsule dosage form ofclaim 29, wherein the dosage is useful for treating or amelioratingconditions or disorders that would benefit from enhancement of nitricoxide synthase (NOS) activity and subjects suffering from vasculardiseases, ischemic or inflammatory diseases, or insulin resistance. 49.The stable capsule dosage form of claim 29, wherein the dosage is usefulfor treating or ameliorating the symptoms of sickle cell disease,peripheral arterial disease, chronic kidney disease, or hypertension.50. The stable capsule dosage form of claim 29, comprising administeringthe dosage to a mammal with food to increase absorption of thetetrahydrobiopterin dihydrochloride.
 51. The stable capsule dosage formof claim 50, wherein the food is a high-fat, or a high-calorie, or ahigh-fat and high-calorie meal.
 52. The stable capsule dosage form ofclaim 29, wherein at least about 90% of the initial amount of thetetrahydrobiopterin dihydrochloride remains after the capsule dosageform is stored in the container at about 40° C. and about 75% relativehumidity for a period of three months.
 53. The stable capsule dosageform of claim 29, wherein at least about 99% of the initial amount ofthe tetrahydrobiopterin dihydrochloride remains after the capsule dosageform is stored in the container at about 40° C. and about 75% relativehumidity for a period of three months.
 54. The stable capsule dosageform of claim 29, wherein the initial amount of tetrahydrobiopterindihydrochloride remains after the capsule dosage form is stored in thecontainer at about 40° C. and about 75% relative humidity for a periodof six months.
 55. The stable capsule dosage form of claim 29, whereinat least about 85% of the initial amount of the tetrahydrobiopterindihydrochloride dissolves within about 15 minutes after the capsuledosage form is stored in the container at about 40° C. and about 75% RHfor a period from about three months to about six months, and whereinthe dissolution is determined according to U.S.P. Method II at 50 r.p.m.in 0.1N hydrochloric acid maintained at 37° C.
 56. The stable capsuledosage form of claim 29, wherein at least about 90% of the initialamount of the tetrahydrobiopterin dihydrochloride remains, and whereinat least about 85% of the initial amount of the tetrahydrobiopterindihydrochloride dissolves within about 15 minutes, after the capsuledosage form is stored in the container at about 40° C. and about 75% RHfor a period of about three months.
 57. The stable capsule dosage formof claim 29, wherein the stable capsule dosage form is for oraladministration.
 58. A method of treating hyperphenylananinemia due toBH4 deficiency, wherein the hyperphenylalaninemia due to BH4 deficiencyis associated with deficiency in or reduced activity of any one or anycombination of the enzymes GTP cyclohydrolase1,6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase,dihydropteridine reductase, and pterin-4-carbinolamine dehydratase,comprising administering the composition of claim
 1. 59. A method ofreducing blood phenylalanine levels in patients withhyperphenylalaninemia due to tetrahydrobiopterin-responsivephenylketonuria, comprising administering the composition of claim 1.60. A method of reducing blood phenylalanine levels in patients withhyperphenylalaninemia due to tetrahydrobiopterin-responsivephenylketonuria, comprising administering the composition of claim 1.61. A method of treating or ameliorating conditions associated withelevated phenylalanine levels or decreased tyrosine or tryptophanlevels, comprising administering the composition of claim
 1. 62. Amethod of treating or ameliorating autism, comprising administering thecomposition of claim
 1. 63. A method of treating or amelioratingconditions or disorders that would benefit from enhancement of nitricoxide synthase (NOS) activity or subjects suffering from vasculardiseases, ischemic or inflammatory diseases, or insulin resistance,comprising administering the composition of claim
 1. 64. A method oftreating or ameliorating the symptoms of sickle cell disease, peripheralarterial disease, chronic kidney disease, or hypertension, comprisingadministering the composition of claim
 1. 65. A method of administeringthe pharmaceutical composition of claim 1 to a mammal with food toincrease absorption of the BH4 or BH4-related compound.
 66. The methodof claim 65, wherein the food is a high-fat, or a high-calorie, or ahigh-fat and high-calorie meal.
 67. A process for preparing a stable,dry blend powder of claim 1, comprising: blending half of a filler withthe BH4 or BH4-related compound and a flavor enhancer in a blender toachieve an adequate mixture; further blending a portion of the firstblended mixture with acesulfame potassium or sucralose, and ascorbicacid; passing the second mixture through a suitable sieve; and blendingthe remainder of the first mixture with the second mixture to achieve ahomogenous mixture.
 68. A pharmaceutical composition of claim 1,prepared by a process comprising: blending half of a filler with the BH4or BH4-related compound and a flavor enhancer in a blender to achieve anadequate mixture; blending a portion of the first blended mixture withacesulfame potassium or sucralose, the filler, and ascorbic acid;passing the second mixture through a suitable sieve; and blending theremainder of the first mixture with the second mixture to achieve ahomogenous mixture.
 69. The process of claim 67, wherein the sieve is a#20 mesh sieve.
 70. The pharmaceutical composition of claim 68, whereinthe sieve is a #20 mesh sieve.
 71. The process of claim 67, wherein theblender is a V-blender.
 72. The pharmaceutical composition of claim 68,wherein the blender is a V-blender.